Compositions and methods of administering paclitaxel with other drugs using medical devices

ABSTRACT

Systems and compositions comprising paclitaxel and a second drug, such as rapamycin, analogs, derivatives, salts and esters thereof are disclosed, as well as methods of delivery wherein the drugs have effects that complement each other. Medical devices comprising supporting structures capable of including or supporting a pharmaceutically acceptable carrier or excipient, which carrier or excipient can contain one or more therapeutic agents or substances, with the carrier preferably including a coating on the surface thereof, and the coating including the therapeutic substances, such as, for example, drugs. Supporting structures for the medical devices that are suitable for use in this invention include coronary stents, peripheral stents, catheters, arterio-venous grafts, by-pass grafts, and drug delivery balloons used in the vasculature. These compositions and systems can be used in combination with other drugs, including anti-proliferative agents, anti-platelet agents, anti-inflammatory agents, anti-thrombotic agents, cytotoxic drugs, agents that inhibit cytokine or chemokine binding, cell de-differentiation inhibitors, anti-lipaedemic agents, matrix metalloproteinase inhibitors, cytostatic drugs, or combinations of these and other drugs.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a continuation-in-part of U.S. Ser. No. 10/235,572,filed Sep. 6, 2002 now abandoned, which is a continuation in part ofU.S. Ser. No. 09/950,307, filed Sep. 10, 2001, now U.S. Pat. No.6,890,546, which is a continuation-in-part of U.S. Ser. No. 09/433,001,filed Nov. 2, 1999, now U.S. Pat. No. 6,329,386, which is a divisionalof U.S. Ser. No. 09/159,945, filed Sep. 24, 1998, now U.S. Pat. No.6,015,815 and claims priority to U.S. Ser. No. 60/060,105, filed Sep.26, 1997; this application also claims priority to U.S. Ser. No.60/664,328 filed on Mar. 23, 2005, U.S. Ser. No. 60/727,080 filed Oct.14, 2005, U.S. Ser. No. 60/726,878 filed Oct. 14, 2005, U.S. Ser. No.60/732,577 filed Oct. 17, 2005, U.S. Ser. No. 60/554,730 filed Mar. 19,2004 which is a provisional application of U.S. Ser. No. 11/084,172filed Mar. 18, 2005, and U.S. Ser. No. 60/727,196 filed Oct. 14, 2005;the entirety of all the above of which is incorporated herein byreference.

STATEMENT REGARDING FEDERALLY SPONSORED RESEARCH OR DEVELOPMENT

Not Applicable

INCORPORATION-BY-REFERENCE OF MATERIAL SUBMITTED ON A COMPACT DISC

Not Applicable.

TECHNICAL FIELD

The invention relates to novel chemical compounds havingimmunomodulatory activity and/or anti-restenotic activity and syntheticintermediates useful for the preparation of the novel compounds, and inparticular to macrolide immunomodulators. More particularly, theinvention relates to semisynthetic analogs of rapamycin, means for theirpreparation, pharmaceutical compositions including such compounds, andmethods of treatment employing the same.

BACKGROUND OF THE INVENTION

Introduction

The compound cyclosporine (cyclosporin A) has found wide use since itsintroduction in the fields of organ transplantation andimmunomodulation, and has brought about a significant increase in thesuccess rate for transplantation procedures. Recently, several classesof macrocyclic compounds having potent immunomodulatory activity havebeen discovered. Okuhara et al. disclose a number of macrocycliccompounds isolated from the genus Streptomyces, including theimmunosuppressant FK-506, a 23-membered macrocyclic lactone, which wasisolated from a strain of S. tsukubaensis (Okuhara et al., 1986).

Other related natural products, including FR-900520 and FR-900523, whichdiffer from FK-506 in their alkyl substituent at C-21, have beenisolated from S. hygroscopicus yakushimnaensis. Another analog,FR-900525, produced by S. tsukubaensis, differs from FK-506 in thereplacement of a pipecolic acid moiety with a proline group.Unsatisfactory side-effects associated with cyclosporine and FK-506 suchas nephrotoxicity, have led to a continued search for immunosuppressantcompounds having improved efficacy and safety, including animmunosuppressive agent which is effective topically, but ineffectivesystemically (Luly, 1995).

Rapamycin

Rapamycin is a macrocyclic triene antibiotic produced by Streptomyceshygroscopicus, which was found to have antifungal activity, particularlyagainst Candida albicans, both in vitro and in vivo (C. Vezina et al.,J. Antibiot. 1975, 28, 721; S. N. Sehgal et al., J. Antibiot. 1975, 28,727; H. A. Baker et al., J. Antibiot. 1978, 31, 539; U.S. Pat. Nos.3,929,992; and 3,993,749).

Rapamycin alone (Surendra, 1989) or in combination with picibanil (Eng,1983) has been shown to have anti-tumor activity. In 1977, rapamycin wasalso shown to be effective as an immunosuppressant in the experimentalallergic encephalomyelitis model, a model for multiple sclerosis; in theadjuvant arthritis model, a model for rheumatoid arthritis; and wasshown to effectively inhibit the formation of IgE-like antibodies(Martel et al., 1977).

The immunosuppressive effects of rapamycin have also been disclosed inFASEB, 1989, 3, 3411 as has its ability to prolong survival time oforgan grafts in histo-incompatible rodents Morris and Meiser, 1989). Theability of rapamycin to inhibit T-cell activation was disclosed by M.Strauch (FASEB, 1989, 3, 3411). These and other biological effects ofrapamycin have been previously reviewed (Morris, 1992).

Rapamycin has been shown to reduce neointimal proliferation in animalmodels, and to reduce the rate of restenosis in humans. Evidence hasbeen published showing that rapamycin also exhibits an anti-inflammatoryeffect, a characteristic which supported its selection as an agent forthe treatment of rheumatoid arthritis. Because both cell proliferationand inflammation are thought to be causative factors in the formation ofrestenotic lesions after balloon angioplasty and stent placement,rapamycin and analogs thereof have been proposed for the prevention ofrestenosis.

Mono-ester and di-ester derivatives of rapamycin (esterification atpositions 31 and 42) have been shown to be useful as antifungal agents(Rakhit, 1982 U.S. Pat. No. 4,316,885,) and as water soluble prodrugs ofrapamycin (Stella, 1987, U.S. Pat. No. 4,650,803).

Fermentation and purification of rapamycin and 30-demethoxy rapamycinhave been described in the literature (C. Vezina et al. J. Antibiot.Tokyo), 1975, 28 (10), 721; S. N. Sehgal et al., J. Antibiot. Tokyo),1975, 28(10), 727; 1983, 36(4), 351; N. L. Paiva et al., J. NaturalProducts, 1991, 54(1), 167-177).

Numerous chemical modifications of rapamycin have been attempted. Theseinclude the preparation of mono- and di-ester derivatives of rapamycin(Caufield, 1992), 27-oximes of rapamycin (Failli, 1992a); 42-oxo analogof rapamycin (Caufield, 1991); bicyclic rapamycins (Kao, 1992a);rapamycin dimers (Kao, 1992b); silyl ethers of rapamycin (Failli,1992b); and arylsulfonates and sulfamates (Failli, 1993). Rapamycin wasrecently synthesized in its naturally occurring enantiomeric form(Hayward et al., 1993; Nicolaou et al., 1993; Romo et al., 1993).

It has been known that rapamycin, like FK-506, binds to FKBP-12(Siekierka, J. J.; Hung, S. H. Y.; Poe, M.; Lin, C. S.; Sigal, N. H.Nature, 1989, 341, 755-757; Harding, M. W.; Galat, A.; Uehling, D. E.;Schreiber, S. L. Nature 1989, 341, 758-760; Dumont, F. J.; Melino, M.R.; Staruch, M. J.; Koprak, S. L.; Fischer, P. A.; Sigal, N. H. J.Immol. 1990, 144, 1418-1424; Bierer, B. E.; Schreiber, S. L.; Burakoff,S. J. Eur. J. Immunol. 1991, 21, 439-445; Fretz, H.; Albers, M. W.;Galat, A.; Standaert, R. F.; Lane, W. S.; Burakoff, S. J.; Bierer, B.E.; Schreiber, S. L. J. Am. Chem. Soc. 1991, 113, 1409-1411). It hasalso been shown that the rapamycin/FKBP-12 complex binds to yet anotherprotein, m-TOR, which is distinct from calcineurin, the protein that theFK-506/FKBP-12 complex inhibits Brown, E. J.; Albers, M. W.; Shin, T.B.; Ichikawa, K.; Keith, C. T.; Lane, W. S.; Schreiber, S. L. Nature1994, 369, 756-758; Sabatini, D. M.; Erdjument-Bromage, H.; Lui, M.;Tempest, P.; Snyder, S. H. Cell, 1994, 78, 35-43).

Other drugs have been used to counter unwanted cell proliferation.Exemplary of these is paclitaxel. A complex alkaloid extracted from thePacific Yew, Taxus brevifolia, paclitaxel stabilizes components of thecell skeleton (tubulin, the building blocks of microtubules) that arecritical in cell division, thus preventing cell proliferation Miller andOjima, 2001).

Stents

Percutaneous transluminal coronary angioplasty PTCA) was developed byAndreas Gruentzig in the 1970's. The first canine coronary dilation wasperformed on Sep. 24, 1975; studies showing the use of PTCA werepresented at the annual meetings of the American Heart Association thefollowing year. Shortly thereafter, the first human patient was studiedin Zurich, Switzerland, followed by the first American human patients inSan Francisco and New York. While this procedure changed the practice ofinterventional cardiology with respect to treatment of patients withobstructive coronary artery disease, the procedure did not providelong-term solutions. Patients received only temporary abatement of thechest pain associated with vascular occlusion; repeat procedures wereoften necessary. It was determined that the existence of restenoticlesions severely limited the usefulness of the new procedure. In thelate 1980's, stents were introduced to maintain vessel patency afterangioplasty. Stenting is involved in 90% of the angioplasties performedtoday. Before the introduction of stents, the rate of restenosis rangedfrom 30-50% of the patients who were treated with balloon angioplasty.Introduction of stenting resulted in further improvements in outcomes,with restenosis rates of 1530%. Following stenting, the restenosislesion is caused primarily by neointimal hyperplasia, which isdistinctly different from atherosclerotic disease both in time-courseand in histopathologic appearance. Restenosis is a healing process ofdamaged coronary arterial walls, with neointimal tissue impingingsignificantly on the vessel lumen. Vascular brachytherapy appears to beefficacious against in-stent restenosis lesions. Radiation, however, haslimitations of practicality and expense, and lingering questions aboutsafety and durability.

Stents and Combination Therapies

The major effort undertaken by the interventional device community tofabricate and evaluate drug eluting stents has met the original goal byreducing restenosis by at least 50%. However, there still remains a needfor improved local drug delivery devices, e.g., drug-impregnatedpolymer-coated stents that provide safe and efficacious tools forpreventing and treating of restenosis. For example, the two commerciallyavailable single-drug eluting stents reduce restenosis and improvepatient outcomes, but do not eliminate restenosis or are free of adversesafety issues. Patients, and especially at-risk patients, includingdiabetics, those with small vessels and those with acute coronarysyndromes, could benefit from local drug delivery devices, such asstents with improved capabilities.

Drug delivery devices including combinations of drugs are known.However, the art does not appear to teach particularly effective drugcombinations administered locally, e.g., eluted from a stent. Forexample, and as discussed more below, Falotico teaches an EVA-PBMApolymer-coated stent including a rapamycin/dexamethasone combination was“far less effective” in reducing neointimal area, percent-area stenosis,and inflammation scores than stents delivering either rapamycin alone ordexamethasone alone.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 shows blood concentrations±SEM (n=3) of tetrazole-containingrapamycin analogs dosed in monkey.

FIG. 2 is a side view in elevation showing a stent suitable for use inthis invention.

FIG. 3A is a cross-sectional view of a vessel segment in which wasplaced a stent coated with a polymer only.

FIG. 3B is a cross-sectional view of a vessel segment in which wasplaced a stent coated with a polymer plus drug.

FIG. 4 shows on a linear scale mean blood-concentration—time plot forsingle escalating intravenous doses of zotarolimus in humans.

FIG. 5 shows on a log-linear scale mean blood concentration-time plots,following single escalating intravenous doses of zotarolimus in humans.

FIG. 6 shows dose proportionality of zotarolimus C_(max) and AUCparameters following single escalating intravenous doses in humans.

FIG. 7 shows mean blood concentration-time plots of zotarolimusfollowing multiple intravenous doses in humans.

FIG. 8 shows mean zotarolimus blood concentration-time profiles for 200,400 and 800 μg QD (daily) dose groups on Day 1 (FIG. 8 a), Day 14 (FIG.8 b), and Days 1-14 (FIG. 8 c).

FIG. 9 shows observed zotarolimus concentration-time data over days 1through 14 for 800 μg QD dose group.

FIG. 10 shows that tacrolimus blocks the anti-proliferative activity ofzotarolimus in smooth muscle cells in vitro (FIG. 10A). Theanti-proliferative activity of zotarolimus, paclitaxel (P) andcombinations in smooth muscle cells (FIG. 10B) and endothelial cells(FIG. 10C) in vitro are also shown. Proliferation was determined bymeasuring; data are the mean±SEM of 3 experiments, except as noted.FIGS. 10D-G show isobologram analyses of combination anti-proliferativeactivity in smooth muscle cells. The concentrations producing thespecified level of anti-proliferative activity were determined from thedose-response curves generated by non-linear curve fitting of the datameans. FIGS. 10H-K show isobologram analyses of the anti-proliferativeactivity of the combination of zotarolimus and paclitaxel in endothelialcells. The concentrations of compounds producing the specified levels ofactivity were determined from the mean data. FIGS. 10L-M shows acombination index (CI) analysis of the antiproliferative activity ofcombinations of ABT-578 and paclitaxel in hCaSMC and hCaEC. CI levelswere determined from the mean data using the method of Chou and Talalay(Chou and Talalay, 1984).

FIG. 11 shows paclitaxel release from stents loaded with paclitaxel (7μg/mm) alone, paclitaxel (7 μg/mm) and zotarolimus (10 μg/mm);paclitaxel (3.5 μg/mm) and zotarolimus (5 μg/mm); or paclitaxel (1μg/mm) and zotarolimus (10 μg/mm).

FIG. 12 shows percent paclitaxel release from stents loaded withpaclitaxel (7 μg/mm) alone, paclitaxel (7 μg/mm) and zotarolimus (10μg/mm); paclitaxel (3.5 μg/mm) and zotarolimus (5 μg/mm); or paclitaxel(1 μg/mm) and zotarolimus (10 μg/mm).

FIG. 13 shows zotarolimus release from stents loaded with zotarolimus(10 μg/mm) alone, paclitaxel (7 μg/mm) and zotarolimus (10 μg/mm);paclitaxel (3.5 μg/mm) and zotarolimus (5 μg/mm); or paclitaxel (1μg/mm) and zotarolimus (10 μg/mm).

FIG. 14 shows the neointimal areas (30% overstretch) after 28 days ofimplantation in swine blood vessels of drug-eluting (single andmultiple) and non-drug-eluting stents; boxed numbers indicate the numberof stents per group.

FIG. 15 shows neointimal thicknesses (30% overstretch) after 28 days ofimplantation in swine blood vessels of drug-eluting (single andmultiple) and non-drug-eluting stents; boxed numbers indicate the numberof stents per group.

FIG. 16 shows percent area stenoses (30% overstretch) after 28 days ofimplantation in swine blood vessels of drug-eluting (single andmultiple) and non-drug-eluting stents; boxed numbers indicate the numberof stents per group.

SUMMARY OF THE INVENTION

In one aspect, embodiments of the invention are directed to a drugdelivery system that has a supporting structure capable of carrying apharmaceutically acceptable carrier or excipient, and a therapeuticcomposition having a taxane or derivatives, prodrugs, or salts thereof,and a second drug or derivatives, prodrugs, or salts thereof, whereinthe formation of neointimal hyperplasia is reduced when the system isimplanted in a lumen of a blood vessel of a subject when compared to acontrol system. The subject can be, for example, a human or a pig. Theratio of paclitaxel:second drug, r, are by weight 10:0.01≧r≧0.01:10, andin some cases, r=1:10. The drug delivery system can include a stent, andcan include a third—or more—drugs or other therapeutic substances,including biologicals. Other therapeutic substances include, but are notlimited to, anti-proliferative agents, anti-platelet agents, steroidaland non-steroidal anti-inflammatory agents, anti-lipidemic agents,anti-thrombotic agents, thrombolytic agents. The subjects can be mammalsincluding, but not limited to, humans and swine.

Another object of embodiments of the invention is directed to a systemfor providing controlled release delivery of drugs for treating orinhibiting neointimal hyperplasia in a blood vessel. The system includespaclitaxel or salts, prodrugs, or derivatives thereof; and a second drugor salts, prodrugs, or derivatives thereof, wherein paclitaxelcomplements activity of the second drug, and wherein the second drugcomplements activity of paclitaxel. The ratio of paclitaxel:second drug,r, are by weight 10:0.01≧r≧0.01:10, and in some cases, r=1:10. The drugdelivery system can include a stent, and can include a third—ormore—drugs or other therapeutic substances, including biologicals. Othertherapeutic substances include, but are not limited to,anti-proliferative agents, anti-platelet agents, steroidal andnon-steroidal anti-inflammatory agents, anti-lipidemic agents,anti-thrombotic agents, thrombolytic agents. The subjects can be mammalsincluding, but not limited to, humans and swine.

A further object of embodiments of the invention is directed topharmaceutical compositions that include paclitaxel or salts, prodrugs,or derivatives thereof; and a second drug or salts, or derivativesthereof, wherein the ratio of paclitaxel:second drug, r, is by weight10:00.1≧r≧0.01:10; wherein if the composition is administered to asubject in a blood vessel on a medical device, and wherein the formationof neointimal hyperplasia is reduced when the system is implanted in alumen of a blood vessel of a subject when compared to a control system.The ratio can be r=1:10, and the formulation is further associated witha medical device, include a stent, or a coated stent. The subjects canbe mammals including, but not limited to, humans and swine. In anotheraspect, the invention is directed to methods of treating subjects, byplacing or administering any of the described systems or compositionsthat include paclitaxel or salts, prodrugs, or derivatives thereof; anda second drug or salts, prodrugs, or derivatives thereof; wherein theratio of paclitaxel:second drug, r, is by weight 10:0.01≧r≧0.01:10.

In another aspect, the invention is directed to kits containing any ofthe described systems or compositions that include paclitaxel or salts,prodrugs, or derivatives thereof; and a second drug or salts, prodrugs,or derivatives thereof; wherein the ratio of paclitaxel:second drug, r,is by weight 10:0.01≧r≧0.01:10.

In another aspect, the invention is directed to a drug delivery system,that includes a stent comprising a coating on a surface, the coatinghaving a therapeutic composition that includes paclitaxel and a seconddrug, or derivatives, prodrugs, or salts thereof, wherein neointimahyperplasia is reduced by ≧10% when compared to the control drugdelivery system, and wherein the ratio, r, of paclitaxel:second drug byweight is 10:0.01≧r≧0.01:10. The ratio, r, can be r=1:10.

In all aspects of the invention, the second drug can be rapamycin,analogs of rapamycin, and salts, prodrugs, or derivatives of rapamycin.

DETAILED DESCRIPTION EMBODIMENTS OF THE INVENTION

Definitions

The term “prodrug,” as used herein, refers to compounds which arerapidly transformed in vivo to the parent compound of the above formula,for example, by hydrolysis in blood. A thorough discussion is providedHiguchi and Stella (Higuchi and Stella, 1987; Roche, 1987) and Roche(Roche, 1987), both of which are incorporated herein by reference.

The term “associated with,” as used herein, refers to compounds whichcan be in many forms including, but not limited to, mixed, unmixed,microspheres, mixed with the coatings, mixed with the support structure.One skilled in the art would appreciate the variations of interactionsbetween drugs, coatings/drugs, and drugs/coatings/support structures.

The term “pharmaceutically acceptable prodrugs”, as used herein, refersto those prodrugs of the compounds in embodiments of the invention whichare, within the scope of sound medical judgment, suitable for use incontact with the tissues of humans and lower mammals without unduetoxicity, irritation, and allergic response, are commensurate with areasonable benefit/risk ratio, and are effective for their intended use.Other embodiments include pharmaceutically acceptable prodrugs that arederivatized at the C-31 hydroxyl group.

The term “prodrug esters,” as used herein, refers to any of severalester-forming groups that are hydrolyzed under physiological conditions.Examples of prodrug ester groups include acetyl, proprionyl, pivaloyl,pivaloyloxymethyl, acetoxymethyl, phthalidyl, methoxymethyl, indanyl,and the like, as well as ester groups derived from the coupling ofnaturally or unnaturally-occurring amino acids to the C-31 hydroxylgroup of compounds of embodiments of the invention.

R═R¹C(O)R²R³; R¹C(S)R²R³

-   -   Where R¹=O, S    -   R²=nothing, 0, N, S, various alkyl, alkenyl, alkynyl,        heterocycles, aryl    -   R³=nothing, various alkyl, alkenyl, alkynyl, heterocycles, aryl    -   Alkyl, alkenyl, alkynyl, heterocycles, aryl groups can be        substituted or unsubstituted

The term “supporting structure” means a framework that is capable ofincluding or supporting a pharmaceutically acceptable carrier orexcipient, which carrier or excipient may include one or moretherapeutic agents or substances, e.g., one or more drugs and/or othercompounds. The supporting structure is typically formed of metal or apolymeric material. Suitable supporting structures formed of polymericmaterials, including biodegradable polymers, capable of including thetherapeutic agents or substances include, without limitation, thosedisclosed in U.S. Pat. Nos. 6,413,272 and 5,527,337, which areincorporated herein by reference (Igaki, 2002; Stack et al., 1996).

The term “complementary” as used herein, refers to the behaviorexhibited by at least two drugs in combination where their respectivepharmaceutical activities benefit from the combination by; in someinstances having additive activity; in some instances having separate,but beneficial activities aiding in the overall desired pharmacologicaleffect in mammals; and where the combination drugs do not activelyreduce each other's biological activity.

“Subject” means a vertebrate including, but not limited to mammals,including a monkey, dog, cat, rabbit, cow, pig, goat, sheep, horse, rat,mouse, guinea pig, and human.

“Therapeutic substance” means any substance that when administered to asubject appropriately at an appropriate doses, has a beneficial effecton the subject.

Rapamycin, a rapamycin analog, or derivatives, or salts thereof refer toall analogs and derivatives of rapamycin, as well as the salts thereof.Examples include tacrolimus, 1,2,3,4-tetrahydro-rapamycin, and others,include those described by Skotnicki et al. (Skotnicki et al., 1994),incorporated herein by reference.

Methods of Treatment

The compounds of the invention, including but not limited to thosespecified in the examples, possess immunomodulatory activity in mammals(including humans). As immunosuppressants, the compounds of embodimentsof the invention are useful for the treatment and prevention ofimmune-mediated diseases including the resistance by transplantation oforgans or tissue including heart, kidney, liver, medulla ossium, skin,cornea, lung, pancreas, intestinum tenue, limb, muscle, nerves,duodenum, small-bowel, pancreatic-islet-cell, and the like;graft-versus-host diseases brought about by medulla ossiumtransplantation; autoimmune diseases including rheumatoid arthritis,systemic lupus erythematosus, Hashimoto's thyroiditis, multiplesclerosis, myasthenia gravis, Type I diabetes, uveitis, allergicencephalomyelitis, glomerulonephritis, and the like. Further usesinclude the treatment and prophylaxis of inflammatory andhyperproliferative skin diseases and cutaneous manifestations ofimmunologically-mediated illnesses, including psoriasis, atopicdermatitis, contact dermatitis and further eczematous dermatitises,seborrhoeis dermatitis, lichen planus, pemphigus, bullous pemphigoid,epidermolysis bullosa, urticaria, angioedemas, vasculitides, erythemas,cutaneous eosinophilias, lupus erythematosus, acne and alopecia greata;various eye diseases (autoimmune and otherwise) includingkeratoconjunctivitis, vernal conjunctivitis, uveitis associated withBehcet's disease, keratitis, herpetic keratitis, conical cornea,dystrophia epithelialis corneae, corneal leukoma, and ocular pemphigus.In addition reversible obstructive airway disease, including asthma (forexample, bronchial asthma, allergic asthma, intrinsic asthma, extrinsicasthma and dust asthma), particularly chronic or inveterate asthma (forexample, late asthma and airway hyper-responsiveness), bronchitis,allergic rhinitis, and the like are targeted by compounds of thisinvention. Inflammation of mucosa and blood vessels including gastriculcers, vascular damage caused by ischemic diseases and thrombosis.Moreover, hyperproliferative vascular diseases including intimal smoothmuscle cell hyperplasia, restenosis and vascular occlusion, particularlyfollowing biologically- or mechanically-mediated vascular injury, couldbe treated or prevented by the compounds of the invention.

The compounds or drugs described herein can be applied to stents thathave been coated with a polymeric compound. Incorporation of thecompound or drug into the polymeric coating of the stent can be carriedout by dipping the polymer-coated stent into a solution including thecompound or drug for a sufficient period of time (such as, for example,five minutes) and then drying the coated stent, such as, for example, bymeans of air drying for a sufficient period of time (such as, forexample, 30 minutes). Other methods of applying therapeutic substances,including spraying, can be used. The polymer-coated stent including thecompound or drug can then be delivered to the coronary vessel bydeployment from a balloon catheter or via a self expanding stent. Inaddition to stents, other devices that can be used to introduce thedrugs of this invention to the vasculature include, but are not limitedto grafts, catheters, and balloons. In addition, other compounds ordrugs that can be used in lieu of the drugs of this invention include,but are not limited to, A-94507 and SDZ RAD (a.k.a. Everolimus).

The compounds described herein for use in polymer-coated stents can beused in combination with other pharmacological agents. Thepharmacological agents that would, in combination with the compounds ofthis invention, be most effective in preventing restenosis can beclassified into the categories of anti-proliferative agents,anti-platelet agents, anti-inflammatory agents, anti-thrombotic agents,and thrombolytic agents. These classes can be further sub-divided. Forexample, anti-proliferative agents can be anti-mitotic. Anti-mitoticagents inhibit or affect cell division, whereby processes normallyinvolved in cell division do not take place. One sub-class ofanti-mitotic agents includes vinca alkaloids. Representative examples ofvinca alkaloids include, but are not limited to, vincristine,paclitaxel, etoposide, nocodazole, indirubin, and anthracyclinederivatives, such as, for example, daunorubicin, daunomycin, andplicamycin. Other sub-classes of anti-mitotic agents includeanti-mitotic alkylating agents, such as, for example, tauromustine,bofumustine, and fotemustine, and anti-mitotic metabolites, such as, forexample, methotrexate, fluorouracil, 5-bromodeox ridine, 6-azacytidine,and cytarabine. Anti-mitotic alkylating agents affect cell division bycovalently modifying DNA, RNA, or proteins, thereby inhibiting DNAreplication, RNA transcription, RNA translation, protein synthesis, orcombinations of the foregoing.

An example of an anti-mitotic agent includes, but is not limited to,paclitaxel. As used herein, paclitaxel includes the alkaloid itself andnaturally occurring forms and derivatives thereof, as well as syntheticand semi-synthetic forms thereof.

Anti-platelet agents are therapeutic entities that act by (1) inhibitingadhesion of platelets to a surface, typically a thrombogenic surface,(2) inhibiting aggregation of platelets, (3) inhibiting activation ofplatelets, or (4) combinations of the foregoing. Activation of plateletsis a process whereby platelets are converted from a quiescent, restingstate to one in which platelets undergo a number of morphologic changesinduced by contact with a thrombogenic surface. These changes includechanges in the shape of the platelets, accompanied by the formation ofpseudopods, binding to membrane receptors, and secretion of smallmolecules and proteins, such as, for example, ADP and platelet factor 4.Anti-platelet agents that act as inhibitors of adhesion of plateletsinclude, but are not limited to, eptifibatide, tirofiban, RGD(Arg-Gly-Asp)-based peptides that inhibit binding to gpIIbIIIa or αvβ3,antibodies that block binding to gpIIaIIIb or αvβ3, anti-P-selectinantibodies, anti-E-selectin antibodies, compounds that block P-selectinor E-selectin binding to their respective ligands, saratin, and anti-vonWillebrand factor antibodies. Agents that inhibit ADP-mediated plateletaggregation include, but are not limited to, disagregin and cilostazol.

Anti-inflammatory agents can also be used. Examples of these include,but are not limited to, prednisone, dexamethasone, hydrocortisone,estradiol, triamcinolone, mometasone, fluticasone, clobetasol, andnon-steroidal anti-inflammatories, such as, for example, acetaminophen,ibuprofen, naproxen, adalimumab and sulindac. The arachidonatemetabolite prostacyclin or prostacyclin analogs is an example of avasoactive antiproliferative. Other examples of these agents includethose that block cytokine activity or inhibit binding of cytokines orchemokines to the cognate receptors to inhibit pro-inflammatory signalstransduced by the cytokines or the chemokines. Representative examplesof these agents include, but are not limited to, anti-IL1, anti-IL2,anti-IL3, anti-IL4, anti-IL8, anti-IL15, anti-IL18, anti-MCP1,anti-CCR2, anti-GM-CSF, and anti-TNF antibodies.

Anti-thrombotic agents include chemical and biological entities that canintervene at any stage in the coagulation pathway. Examples of specificentities include, but are not limited to, small molecules that inhibitthe activity of factor Xa. In addition, heparinoid-type agents that caninhibit both FXa and thrombin, either directly or indirectly, such as,for example, heparins, heparan sulfate, low molecular weight heparins,such as, for example, the compound having the trademark Clivarin®, andsynthetic oligosaccharides, such as, for example, the compound havingthe trademark Arixtra®. Also included are direct thrombin inhibitors,such as, for example, melagatran, ximelagatran, argatroban, inogatran,and peptidomimetics of binding site of the Phe-Pro-Arg fibrinogensubstrate for thrombin. Another class of anti-thrombotic agents that canbe delivered are factor VII/VIIa inhibitors, such as, for example,anti-factor VII/VIIa antibodies, rNAPc2, and tissue factor pathwayinhibitor (TFPI).

Thrombolytic agents, which may be defined as agents that help degradethrombi (clots), can also be used as adjunctive agents, because theaction of lysing a clot helps to disperse platelets trapped within thefibrin matrix of a thrombus. Representative examples of thrombolyticagents include, but are not limited to, urokinase or recombinanturokinase, pro-urokinase or recombinant pro-urokinase, tissueplasminogen activator or its recombinant form, and streptokinase.

Other drugs that can be used in combination with the compounds of thisinvention are cytotoxic drugs, such as, for example, apoptosis inducers,such as TGF, and topoisomerase inhibitors, including10-hydroxycamptothecin, irinotecan, and doxorubicin. Other classes ofdrugs that can be used in combination with the compounds of thisinvention are drugs that inhibit cell de-differentiation and cytostaticdrugs.

Other agents that can be used in combination with the compounds of thisinvention include anti-lipidemic agents, such as, for example,fenofibrate, matrix metalloproteinase inhibitors, such as, for example,batimistat, antagonists of the endothelin-A receptor, such as, forexample, darusentan, and antagonists of the αvβ3 integrin receptor.

Embodiments of the invention further include a third therapeutic drug orsubstance. When a second drug and/or third therapeutic drug is utilizedit includes, but are not limited to, anti-proliferative agents,anti-platelet agents, anti-inflammatory agents, anti-lipidemic agents,anti-thrombotic agents, thrombolytic agents, their salts, prodrugs, andderivatives or any combination thereof. When a second drug and/or thirdtherapeutic drug is a glucocorticosteriod it includes, but is notlimited to, methylprednisolone, prednisolone, prednisone, triamcinolone,dexamethasone, mometasone, beclomethasone, ciclesonide, bedesonide,triamcinolone, clobetasol, flunisolide, loteprednol, budesonide,fluticasone, their salts, prodrugs, and derivatives or any combinationthereof. When a second drug and/or third therapeutic drug is a steroidhormone it includes, but is not limited to, an estradiol and theirsalts, prodrugs, and derivatives or any combination thereof. Inembodiments, when a second drug and/or third therapeutic drug it can besmall molecules and biologics that reduce inflammatory cytokineactivity. When a second drug and/or third therapeutic drug utilizes ananti-cytokine therapies selected from the group consisting of, but isnot limited to, anti-TNFα therapies, adalimumab, anti-MCP-1 therapies,CCR2 receptor antagonists, anti-IL-18 therapies, anti-IL-1 therapies,and their salts, prodrugs, and derivatives, or any combination thereof.When a said second drug and/or third therapeutic drug utilizes ananti-proliferative agent it includes, but is not limited to, alkylatingagents including cyclophosphamide, chlorambucil, busulfan, carmustineand lomustine, anti-metabolites including methotrexate, fluorouracil,cytarabine, mercaptopurine and pentostatin, vinca alkaloids includingvinblastine and vincristine, antibiotics including doxorubicin,bleomycin and mitomycin, antiproliferatives including cisplatin,procarbazine, etoposide and teniposide, their salts, prodrugs, andderivatives, or any combination thereof. When a second drug and/or thirdtherapeutic drug utilizes an anti-platelet agent it includes, but is notlimited to, glycoprotein IIB/IIIA inhibitors including abciximab,eptifibatide and tirofiban, adenosine reuptake inhibitors includingdipyridamole, ADP inhibitors including clopidogrel and ticlopidine,cyclooxygenase inhibitors including acetylsalicylic acid,phosphodiesterase inhibitors including cilostazol, their salts,prodrugs, and derivatives, or any combination thereof. When a seconddrug and/or third therapeutic drug utilizes an anti-inflammatory agentit includes, but is not limited to, steroids including dexamethasone,hydrocortisone, fluticasone, clobetasol, mometasone and estradiol, andnon-steroidal anti-inflammatory agents including acetaminophen,ibuprofen, naproxen, sulindac, piroxicam, mefanamic acid, those thatinhibit binding of cytokines or chemokines to receptors to inhibitpro-inflammatory signals, including antibodies to IL-1, IL-2, IL-8,IL-15, IL-18 and TNF, their salts, prodrugs, and derivatives, or anycombination thereof. When a second drug and/or third therapeutic drugutilizes an anti-thrombotic agent it includes, but is not limited to,heparins including unfractionated heparins and low-molecular weightheparins including clivarin, dalteparin, enoxaparin, nadroparin andtinzaparin, direct thrombin inhibitors including argatroban, hirudin,hirulog, hirugen, their salts, prodrugs, and derivatives, or anycombination thereof. When a second drug and/or third therapeutic drugutilizes an anti-lipidemic agent selected from the group consisting ofnicotinic acid, probucol, HMG CoA reductase inhibitors includingmevastatin, lovastatin, simvastatin, pravastatin, fluvastatin, fibricacid derivatives including fenofibrate, clofibrate, gemfibrozil,lipid-lowering agents including their salts, prodrugs, and derivativesor any combination thereof. When a second drug and/or third therapeuticdrug utilizes thrombolytic agents it includes, but is not limited to,streptokinase, urokinase, pro-urokinase, tissue plasminogen activatorsincluding alteplase, reteplase, tenectaplase, their salts, prodrugs, andderivatives, or any combination thereof.

Polymers

When used in the invention, the coating can comprise any polymericmaterial in which the therapeutic agents, i.e., the drugs, aresubstantially soluble or effectively dispersed. The purpose of thecoating is to serve as a controlled release vehicle for the therapeuticagent or as a reservoir for a therapeutic agent to be delivered at thesite of a lesion. The coating can be polymeric and can further behydrophilic, hydrophobic, biodegradable, or non-biodegradable. Thematerial for the polymeric coating can be selected from the groupconsisting of polycarboxylic acids, cellulosic polymers, gelatin,polyvinylpyrrolidone, maleic anhydride polymers, polyamides, polyvinylalcohols, polyethylene oxides, glycosaminoglycans, polysaccharides,polyesters, polyurethanes, silicones, polyorthoesters, polyanhydrides,polycarbonates, polypropylenes, polylactic acids, polyglycolic acids,polycaprolactones, polyhydroxybutyrate valerates, polyacrylamides,polyethers, and mixtures and copolymers of the foregoing. Coatingsprepared from polymeric dispersions including polyurethane dispersions(BAYHYDROL, etc.) and acrylic acid latex dispersions can also be usedwith the therapeutic agents of the invention.

Biodegradable polymers that can be used in this invention includepolymers including poly(L-lactic acid), poly(DL-lactic acid),polycaprolactone, poly hydroxy butyrate), polyglycolide,poly(diaxanone), poly hydroxy valerate), polyorthoester; copolymersincluding poly (lactide-co-glycolide), polyhydroxy(butyrate-co-valerate), polyglycolide-co-trimethylene carbonate;polyanhydrides; polyphosphoester; polyphosphoester-urethane; polyaminoacids; polycyanoacrylates; biomolecules including fibrin, fibrinogen,cellulose, starch, collagen and hyaluronic acid; and mixtures of theforegoing. Biostable materials that are suitable for use in thisinvention include polymers including polyurethane, silicones,polyesters, polyolefins, polyamides, polycaprolactam, polyimide,polyvinyl chloride, polyvinyl methyl ether, polyvinyl alcohol, acrylicpolymers and copolymers, polyacrylonitrile, polystyrene copolymers ofvinyl monomers with olefins (including styrene acrylonitrile copolymers,ethylene methyl methacrylate copolymers, ethylene vinyl acetate),polyethers, rayons, cellulosics (including cellulose acetate, cellulosenitrate, cellulose propionate, etc.), parylene and derivatives thereof;and mixtures and copolymers of the foregoing.

In some embodiments, the polymers include, but are not limited to,poly(acrylates) such as poly(ethyl methacrylate), poly(n-propylmethacrylate), poly(isopropyl methacrylate), poly(isobutylmethacrylate), poly(sec-butyl methacrylate), poly(n-butyl methacrylate),poly(2-ethylhexyl methacrylate), poly(n-hexyl methacrylate),poly(cyclohexyl methacrylate), poly(n-hexyl methacrylate),poly(isobornyl methacrylate), and poly(trimethylcyclohexylmethacrylate), poly(methyl acrylate), poly(ethyl arylate), poly(n-propylacrylate), poly(isopropyl acrylate), poly(n-butyl acrylate),poly(isobutyl acrylate), poly(sec-butyl acrylate), poly(pentylacrylate), poly(n-hexyl acrylate), poly(cyclohexyl acrylate) and anyderivatives, analogs, homologues, congeners, salts, copolymers andcombinations thereof.

In some embodiments, the polymers include, but are not limited to,poly(ester urethanes), poly(ether urethanes), poly(urea urethanes),poly(urethanes); silicones; fluorosilicones, poly(esters);poly(ethylene); polypropylene); poly(olefins); copolymers ofpoly(isobutylene); triblock copolymers of styrene and isobutylene;triblock copolymers of styrene and ethylene/butylenes; triblockcopolymers of styrene and butadiene; copolymers of ethylene-alphaolefin;vinyl halide polymers and copolymers such as poly(vinyl chloride) andpoly(vinyl fluoride); poly(vinylidene halides) such as, for example,poly(vinylidene chloride) and poly(vinylidene fluoride); poly(vinylidenefluoride-co-hexafluoropropylene), poly(tetrafluoroethylene);poly(tetrafluoroethylene-co-chlorotrifluoroethylene); poly(vinyl ethers)such as, for example, poly(vinyl methyl ether); poly(acrylonitrile);poly(vinyl ketones); poly(vinyl aromatics) such as poly(styrene);poly(vinyl esters) such as poly(vinyl acetate); copolymers of vinylmonomers and olefins such as copolymers of methacrylic acid; copolymersof acrylic acid; copolymers of N-vinyl pyrrolidone; poly(vinylalcohols); poly(ethylene-co-vinyl alcohol) (EVAL), poly(cyanoacrylates);poly(maleic anhydride) and copolymers of maleic anhydride; copolymers ofacrylonitrile-styrene, ABS resins, and copolymers of ethylene-vinylacetate; and any derivatives, analogs, homologues, congeners, salts,copolymers and combinations thereof.

In some embodiments, the polymers include, but are not limited to,poly(amides) such as Nylon 66 and poly(caprolactam); alkyd resins;poly(carbonates); poly(sulfone); poly(oxymethylenes); poly(imides);poly(ester amides); poly(ethers) including poly(alkylene glycols) suchas, for example, poly(ethylene glycol) and polypropylene glycol); epoxyresins; rayon; rayon-triacetate; biomolecules such as, for example,fibrin, fibrinogen, starch, poly(amino acids); peptides, proteins,gelatin, chondroitin sulfate, dermatan sulfate (a copolymer ofD-glucuronic acid or L-iduronic acid and N-acetyl-D-galactosamine),collagen, hyaluronic acid, and glycosaminoglycans; other polysaccharidessuch as, for example, poly(N-acetylglucosamine), chitin, chitosan,cellulose, cellulose acetate, cellulose butyrate, cellulose acetatebutyrate, cellophane, cellulose nitrate, cellulose propionate, celluloseethers, and carboxymethylcellulose; and any derivatives, analogs,homologues, congeners, salts, copolymers and combinations thereof.

Another polymer that can be used in this invention is polyMPC_(w):LAM_(x):HPMA_(y):TSMA_(z)) where w, x, y, and z represent themolar ratios of monomers used in the feed for preparing the polymer andMPC represents the unit 2-methacryoyloxyethylphosphorylcholine, LMArepresents the unit lauryl methacrylate, HPMA represents the unit2-hydroxypropyl methacrylate, and TSMA represents the unit3-trimethoxysilylpropyl methacrylate. The drug-impregnated stent can beused to maintain patency of a coronary artery previously occluded bythrombus and/or atherosclerotic plaque. The delivery of ananti-proliferative agent reduces the rate of in-stent restenosis.Polymers which can be used in this invention include zwitterionicpolymers including phosphorylcholine units.

Other treatable conditions include but are not limited to ischemic boweldiseases, inflammatory bowel diseases, necrotizing enterocolitis,intestinal inflammations/allergies including Coeliac diseases,proctitis, eosinophilic gastroenteritis, mastocytosis, Crohn's diseaseand ulcerative colitis; nervous diseases including multiple myositis,Guillain-Barre syndrome, Meniere's disease, polyneuritis, multipleneuritis, mononeuritis and radiculopathy; endocrine diseases includinghyperthyroidism and Basedow's disease; hematic diseases including purered cell aplasia, aplastic anemia, hypoplastic anemia, idiopathicthrombocytopenic purpura, autoimmune hemolytic anemia, agranulocytosis,pernicious anemia, megaloblastic anemia and anerythroplasia; bonediseases including osteoporosis; respiratory diseases includingsarcoidosis, fibroid lung and idiopathic interstitial pneumonia; skindisease including dermatomyositis, leukoderma vulgaris, ichthyosisvulgaris, photoallergic sensitivity and cutaneous T cell lymphoma;circulatory diseases including arteriosclerosis, atherosclerosis,aortitis syndrome, polyarteritis nodosa and myocardosis; collagendiseases including scleroderma, Wegener's granuloma and Sjogren'ssyndrome; adiposis; eosinophilic fasciitis; periodontal diseaseincluding lesions of gingiva, periodontium, alveolar bone and substantiaossea dentis; nephrotic syndrome including glomerulonephritis; malepattern alopecia or alopecia senilis by preventing epilation orproviding hair germination and/or promoting hair generation and hairgrowth; muscular dystrophy; Pyoderma and Sezary's syndrome; Addison'sdisease; active oxygen-mediated diseases, as for example organ injuryincluding ischemia-reperfusion injury of organs (including heart, liver,kidney and digestive tract) which occurs upon preservation,transplantation or ischemic disease (for example, thrombosis and cardiacinfarction); intestinal diseases including endotoxin-shock,pseudomembranous colitis and colitis caused by drug or radiation; renaldiseases including ischemic acute renal insufficiency and chronic renalinsufficiency; pulmonary diseases including toxinosis caused bylung-oxygen or drug (for example, paracort and bleomycins), lung cancerand pulmonary emphysema; ocular diseases including cataracts, siderosis,retinitis, pigmentosa, senile macular degeneration, vitreal scarring andcorneal alkali burn; dermatitis including erythema multiforme, linearIgA ballous dermatitis and cement dermatitis; and others includinggingivitis, periodontitis, sepsis, pancreatitis, diseases caused byenvironmental pollution (for example, air pollution), aging,carcinogenesis, metastasis of carcinoma and hypobaropathy; diseasescaused by histamine or leukotriene-C₄ release; Behcet's diseaseincluding intestinal-, vasculo- or neuro-Behcet's disease, and alsoBehcet's which affects the oral cavity, skin, eye, vulva, articulation,epididymis, lung, kidney and so on. Furthermore, the compounds of theinvention are useful for the treatment and prevention of hepatic diseaseincluding immunogenic diseases (for example, chronic autoimmune liverdiseases including autoimmune hepatitis, primary biliary cirrhosis andsclerosing cholangitis), partial liver resection, acute liver necrosis(e.g., necrosis caused by toxin, viral hepatitis, shock or anoxia),B-virus hepatitis, non-A/non-B hepatitis, cirrhosis (including alcoholiccirrhosis) and hepatic failure including fulminant hepatic failure,late-onset hepatic failure and “acute-on-chronic” liver failure (acuteliver failure on chronic liver diseases), and moreover are useful forvarious diseases because of their potentially useful activity inaugmention of the primary chemotherapeutic, antiviral,anti-inflammatory, and cardiotonic effects of drugs the patient mayalready be taking.

The ability of the compounds of the invention to treat proliferativediseases can be demonstrated according to previously described methodsin Bunchman E T and C A Brookshire, Transplantation Proceed. 23 967-968(1991); Yamagishi, et al., Biochem. Biophys. Res. Comm. 191 840-846(1993); and Shichiri, et al., J. Clin. Invest. 87 1867-1871 (1991).Proliferative diseases include smooth muscle proliferation, systemicsclerosis, cirrhosis of the liver, adult respiratory distress syndrome,idiopathic cardiomyopathy, lupus erythematosus, diabetic retinopathy orother retinopathies, psoriasis, sclero derma, pro static hyperplasia,cardiac hyperplasia, restenosis following arterial injury or otherpathologic stenosis of blood vessels. In addition, these compoundsantagonize cellular responses to several growth factors, and thereforepossess antiangiogenic properties, making them useful agents to controlor reverse the growth of certain tumors, as well as fibrotic diseases ofthe lung, liver, and kidney.

Aqueous liquid compositions of embodiments of the invention areparticularly useful for the treatment and prevention of various diseasesof the eye including autoimmune diseases (including, for example,conical cornea, keratitis, dysophia epithelialis corneae, leukoma,Mooren's ulcer, sclevitis and Graves' opthalmopathy) and rejection ofcorneal transplantation.

When used in the above or other treatments, a therapeutically effectiveamount of one of the compounds of the embodiments of the invention maybe employed in pure form or, where such forms exist, in pharmaceuticallyacceptable salt, ester or prodrug form. Alternatively, the compound maybe administered as a pharmaceutical composition including the compoundof interest in combination with one or more pharmaceutically acceptableexcipients. The phrase “therapeutically effective amount” of thecompound of the invention means a sufficient amount of the compound totreat disorders, at a reasonable benefit/risk ratio applicable to anymedical treatment. It will be understood, however, that the total dailyusage of the compounds and compositions of the embodiments of theinvention will be decided by the attending physician within the scope ofsound medical judgment. The specific therapeutically effective doselevel for any particular patient will depend upon a variety of factorsincluding the disorder being treated and the severity of the disorder;activity of the specific compound employed; the specific compositionemployed; the age, body weight, general health, sex and diet of thepatient; the time of administration, route of administration, and rateof excretion of the specific compound employed; the duration of thetreatment; drugs used in combination or coincidental with the specificcompound employed; and like factors well known in the medical arts. Forexample, it is well within the skill of the art to start doses of thecompound at levels lower than required to achieve the desiredtherapeutic effect and to gradually increase the dosage until thedesired effect is achieved.

The total daily dose of the compounds in embodiments of this inventionadministered to a human or lower animal may range from about 0.01 toabout 10 mg/kg/day. For purposes of oral administration, doses may be inthe range of from about 0.001 to about 3 mg/kg/day. For the purposes oflocal delivery from a stent, the daily dose that a patient will receivedepends on the length of the stent. For example, a 15 mm coronary stentmay include a drug in an amount ranging from about 1 to about 600micrograms and may deliver that drug over a time period ranging fromseveral hours to several weeks. When desired, the effective daily dosemay be divided into multiple doses for purposes of administration;consequently, single dose compositions may include such amounts orsubmultiples thereof to make up the daily dose. One skilled in the artcould use the invention for topical administration and doses woulddepend on the site of application.

Within the scope of the invention, there is much flexibility inproviding suitable drug-loaded polymer layers. For example, withintherapeutic window parameters (generally levels between therapeuticallyeffective and toxicity) associated with the drugs of interest, ratios ofthe drugs used in combination can be varied relative to each other. Forexample, an embodiment has a 90:10 total drug:polymer ratio where theratio of drugs in the combination can be 1:1. Thus, a stent delivering azotarolimus/dexamethasone combination according to the invention caninclude 10 mcg/mm zotarolimus and 10 mcg/mm dexamethasone in a PCpolymer layer with a 5 mcg/mm PC topcoat. Total drug:polymer ratio canbe lower, however, e.g., 40:60 or less. Upper limits on the total amountof drug will depend on several factors, including miscibility of theselected drugs in the selected polymer, the stability of thedrug/polymer mixture, e.g., compatibility with sterilization, and thephysical properties of the mixture, e.g., flowability/processability,elasticity, brittleness, viscosity (does not web or bridge between stentstruts), coating thickness that adds substantially to the stent profileor causes delamination or cracking or is difficult to crimp. Embodimentsof the invention include stent struts spaced about 60-80 microns apart,suggesting an upper limit in thickness of the drug/polymer/polymerovercoat is about 30 microns; however, any stent size, strut size andspatial spacing, and/or stent construction can be utilized for drugdelivery as described therein. In embodiments, the therapeutic amount ofan olimus drug includes zotarolimus or everolimus and is at least 1μg/mm stent. In other embodiments, the second drug is aglucocorticosteriod. When the second drug is utilized in embodiments,this second drug is dexamethasone and the therapeutic amount is at least0.5 μg/mm stent

Overcoat thickness (if an overcoat is used) desirably should not undulyimpede release kinetics of the drugs. The overcoat can also be loadedwith one or more drugs, which can be the same or different than those inthe underlying drug-loaded polymer layer.

Generally speaking, drugs useful in combinations for the invention willnot adversely affect the desired activity of the other drug in thecombination. The drugs proposed for use in the combination may be havecomplementary activities or mechanisms of action. Thus, one drug in theproposed combination should not inhibit the desired activity, e.g.,anti-proliferative activity, of the other drug. Nor should either drugcause or enhance the degradation of the other drug. However, a drug thatmight otherwise appear to be unsuitable because, for example, itdegrades during sterilization, can in fact be useful because of aninteraction by another drug. Thus, dexamethasone, which alone has beenobserved to degrade during EtO sterilization, can be used successfullyin combination with zotarolimus, due to the hydrophobicity ofzotarolimus. Moreover, zotarolimus has been observed to reduce theelution rate of dexamethasone, as described in Applicants' co-pendingU.S. patent application Ser. No. 10/796,423.

The pharmaceutical compositions of embodiments of the invention comprisea compound and a pharmaceutically acceptable carrier or excipient, whichmay be administered orally, rectally, parenterally, intracisternally,intravaginally, intraperitoneally, topically (as by powders, ointments,drops or transdermal patch), bucally, as an oral or nasal spray, orlocally as in a stent placed within the vasculature as in a ballooncatheter, or delivery to the pericardial space or into or onto themyocardium. The phrase “pharmaceutically acceptable carrier” means anon-toxic solid, semi-solid or liquid filler, diluent, encapsulatingmaterial or formulation auxiliary of any type. The term “parenteral,” asused herein, refers to all modes of administration other than oral,which include, but not limited to, intravenous, intraarterial,intramuscular, intraperitoneal, intrasternal, subcutaneous andintraarticular injection, infusion, transdermal, and placement, such as,for example, in the vasculature.

Pharmaceutical compositions of this invention for parenteral injectioncomprise pharmaceutically acceptable sterile aqueous or nonaqueoussolutions, dispersions, suspensions, nanoparticle suspensions, oremulsions as well as sterile powders for reconstitution into sterileinjectable solutions or dispersions just prior to use. Examples ofsuitable aqueous and nonaqueous carriers, diluents, solvents or vehiclesinclude water, ethanol, polyols (including glycerol, propylene glycol,polyethylene glycol, and the like), carboxymethylcellulose and suitablemixtures thereof, vegetable oils (including olive oil), and injectableorganic esters including ethyl oleate. Proper fluidity can bemaintained, for example, by the use of coating materials includinglecithin, by the maintenance of the required particle size in the caseof dispersions, and by the use of surfactants.

These compositions may also include adjuvants such as, for example,preservatives, wetting agents, emulsifying agents, and dispersingagents. Prevention of the action of microorganisms may be ensured by theinclusion of various antibacterial and antifungal agents, for example,paraben, chlorobutanol, phenol sorbic acid, and the like. It may also bedesirable to include isotonic agents including sugars, sodium chloride,and the like. Prolonged absorption of the injectable pharmaceutical formmay be brought about by the inclusion of agents that delay absorptionincluding aluminum monostearate and gelatin.

In some cases, in order to prolong the effect of the drug, it isdesirable to slow the absorption of the drug from subcutaneous orintramuscular injection. This may be accomplished by the use of a liquidsuspension of crystalline or amorphous material with poor watersolubility. The rate of absorption of the drug then depends upon itsrate of dissolution which, in turn, may depend upon crystal size andcrystalline form. Alternatively, delayed absorption of a parenterallyadministered drug form is accomplished by dissolving or suspending thedrug in an oil vehicle.

Injectable depot forms are made by forming microencapsule matrices ofthe drug in biodegradable polymers including polylactide-polyglycolide.Depending upon the ratio of drug to polymer and the nature of theparticular polymer employed, the rate of drug release can be controlled.Examples of other biodegradable polymers include poly(orthoesters) andpoly(anhydrides). Depot injectable formulations are also prepared byentrapping the drug in liposomes or microemulsions which are compatiblewith body tissues.

The injectable formulations can be sterilized, for example, byfiltration through a bacterial-retaining filter, or by incorporatingsterilizing agents in the form of sterile solid compositions which canbe dissolved or dispersed in sterile water or other sterile injectablemedium just prior to use.

Solid dosage forms for oral administration include capsules, tablets,pills, powders, and granules. In such solid dosage forms, the activecompound is mixed with at least one inert, pharmaceutically acceptableexcipient or carrier including sodium citrate or dicalcium phosphateand/or a) fillers or extenders including starches, lactose, sucrose,glucose, mannitol, and silicic acid, b) binders such as, for example,carboxymethylcellulose, alginates, gelatin, polyvinylpyrrolidone,sucrose, and acacia, c) humectants including glycerol, d) disintegratingagents including agar-agar, calcium carbonate, potato or tapioca starch,alginic acid, certain silicates, and sodium carbonate, e) solutionretarding agents including paraffin, f absorption accelerators includingquaternary ammonium compounds, g) wetting agents such as, for example,cetyl alcohol and glycerol monostearate, h) absorbents including kaolinand bentonite clay, and i) lubricants including talc, calcium stearate,magnesium stearate, solid polyethylene glycols, sodium lauryl sulfate,and mixtures thereof. In the case of capsules, tablets and pills, thedosage form may also comprise buffering agents.

Solid compositions of a similar type may also be employed as fillers insoft, semi-solid and hard-filled gelatin capsules or liquid-filledcapsules using such excipients as lactose or milk sugar as well as highmolecular weight polyethylene glycols and the like.

The solid dosage forms for oral administration, not limited to, tablets,dragees, capsules, pills, and granules can be prepared with coatings andshells including enteric coatings and other coatings well known in thepharmaceutical formulating art. They may optionally contain opacifyingagents and can also be of a composition that they release the activeingredient(s) only, or preferentially, in a certain part of theintestinal tract, optionally, in a delayed manner. Examples of embeddingcompositions that can be used include polymeric substances and waxes.Those embedding compositions including a drug can be placed on medicaldevices, including stents, grafts, catheters, and balloons.

The active compounds can also be in micro-encapsulated form, ifappropriate, with one or more of the above-mentioned excipients.

Liquid dosage forms for oral administration include pharmaceuticallyacceptable emulsions, solutions, suspensions, syrups and elixirs. Inaddition to the active compounds, the liquid dosage forms may includeinert diluents commonly used in the art such as, for example, water orother solvents, solubilizing agents and emulsifiers including ethylalcohol, isopropyl alcohol, ethyl carbonate, ethyl acetate, benzylalcohol, benzyl benzoate, propylene glycol, 1,3-butylene glycol,dimethyl formamide, oils (in particular, cottonseed, groundnut, corn,germ, olive, castor, and sesame oils), glycerol, tetrahydrofurfurylalcohol, polyethylene glycols and fatty acid esters of sorbitan, andmixtures thereof.

Besides inert diluents, the oral compositions can also include adjuvantsincluding wetting agents, emulsifying and suspending agents, sweetening,flavoring, and perfuming agents.

Suspensions, in addition to the active compounds, may include suspendingagents such as, for example, ethoxylated isostearyl alcohols,polyoxyethylene sorbitol and sorbitan esters, microcrystallinecellulose, aluminum metahydroxide, bentonite, agar-agar, and tragacanth,and mixtures thereof.

Topical administration includes administration to the skin includingsurfaces of the eye. Compositions for topical use on the skin alsoinclude ointments, creams, lotions, and gels. A further form of topicaladministration is to the eye, as for the treatment of immune-mediatedconditions of the eye including autoimmune diseases, allergic orinflammatory conditions, and corneal transplants. The compound of theinvention is delivered in a pharmaceutically acceptable ophthalmicvehicle, such that the compound is maintained in contact with the ocularsurface for a sufficient time period to allow the compound to penetratethe corneal and internal regions of the eye, as for example the anteriorchamber, posterior chamber, vitreous body, aqueous humor, vitreoushumor, cornea, iris/cilary, lens, choroid/retina and sclera. Thepharmaceutically acceptable ophthalmic vehicle may, for example, be anointment, vegetable oil or an encapsulating material.

Compositions for mucosal administration, specially those for inhalation,may be prepared as a dry powder which may be pressurized ornon-pressurized. In non-pressurized powder compositions, the activeingredient in finely divided form may be used in admixture with alarger-sized pharmaceutically acceptable inert carrier comprisingparticles having a size, for example, of up to 100 micrometers indiameter. Suitable inert carriers include sugars including lactose.Desirably, at least 95% by weight of the particles of the activeingredient have an effective particle size in the range of 0.01 to 10micrometers. In rectal or vaginal transmucosal administrationformulations include suppositories or retention enemas which can beprepared by mixing the compounds of this invention with suitablenon-irritating excipients or carriers including cocoa butter,polyethylene glycol or a suppository wax which are solid at roomtemperature but liquid at body temperature and therefore melt in therectum or vaginal cavity and release the active compound.

Alternatively, the composition may be pressurized and include acompressed gas, including nitrogen or a liquefied gas propellant. Theliquefied propellant medium and indeed the total composition is suchthat the active ingredient does not dissolve therein to any substantialextent. The pressurized composition may also include a surface activeagent. The surface active agent may be a liquid or solid non-ionicsurface active agent or may be a solid anionic surface active agent. Inother embodiments, the use of the solid anionic surface active agent isin the form of a sodium salt.

Compounds of embodiments of the invention can also be administered inthe form of liposomes. As is known in the art, liposomes are generallyderived from phospholipids or other lipid substances. Liposomes areformed by mono- or multi-lamellar hydrated liquid crystals that aredispersed in an aqueous medium. Any non-toxic, physiologicallyacceptable and metabolizable lipid capable of forming liposomes can beused. Composition embodiments in liposome form can include, in additionto a compound of the invention, stabilizers, preservatives, excipients,and the like. Lipids in embodiments are the phospholipids and thephosphatidyl cholines (lecithins), both natural and synthetic. Methodsto form liposomes are known in the art. See, for example, Prescott, Ed.,Methods in Cell Biology, Volume XIV, Academic Press, New York, N.Y.(1976), p. 33 et seq.

Compounds of embodiments of the invention may also be coadministeredwith one or more systemic immunosuppressant agents. Theimmunosuppressant agents within the scope of this invention include, butare not limited to, IMURAN® azathioprine sodium, brequinar sodium,SPANIDIN® gusperimus trihydrochloride (also known as deoxyspergualin),mizoribine (also known as bredinin), CELLCEPT® mycophenolate mofetil,NEORAL® Cylosporin A (also marketed as different formulation ofCyclosporin A under the trademark SANDIMMUNE®), PROGRAF® tacrolimus(also known as FK-506), sirolimus and RAPAMUNE®, everolimus, leflunomide(also known as HWA-486), glucocorticoids, including prednisolone and itsderivatives, antibody therapies including orthoclone (OKT3) andZenapax®, leukemia therapies, and antithymyocyte globulins, includingthymoglobulins.

Embodiments

Preparation of Compounds of this Invention

The compounds and processes of embodiments of the invention will bebetter understood in connection with the following synthetic schemeswhich illustrate the methods by which the compounds of the invention maybe prepared.

The compounds of this invention may be prepared by a variety ofsynthetic routes. A representative procedure is shown in Scheme 1.

As shown in Scheme 1, conversion of the C-42 hydroxyl of rapamycin to atrifluoromethanesulfonate or fluorosulfonate leaving group provided A.Displacement of the leaving group with tetrazole in the presence of ahindered, non-nucleophilic base, including 2,6-lutidine,diisopropylethyl amine provided isomers B and C, which were separatedand purified by flash column chromatography.

Synthetic Methods

The foregoing may be better understood by reference to the followingexamples which illustrate the methods by which the compounds of theinvention may be prepared and are not intended to limit the scope of theinvention as defined in the appended claims.

EXAMPLE 1 42(2-tetrazolyl)-rapamycin (Less Polar Isomer) EXAMPLE 1A

A solution of rapamycin (100 mg, 0.11 mmol) in dichloromethane (0.6 mL)at −78° C. under a nitrogen atmosphere was treated sequentially with2,6-lutidine (53 uL, 0.46 mmol, 4.3 eq.) and trifluoromethanesulfonicanhydride (37 uL, 0.22 mmol), and stirred thereafter for 15 minutes,warmed to room temperature and eluted through a pad of silica gel (6 mL)with diethyl ether. Fractions including the triflate were pooled andconcentrated to provide the designated compound as an amber foam.

EXAMPLE 1B 42 (2-tetrazolyl)-rapamycin (Less Polar Isomer)

A solution of Example 1A in isopropyl acetate (0.3 mL) was treatedsequentially with diisopropylethylamine (87 mL, 0.5 mmol) and1H-tetrazole (35 mg, 0.5 mmol), and thereafter stirred for 18 hours.This mixture was partitioned between water (10 mL) and ether (10 mL).The organics were washed with brine (10 mL) and dried Na₂SO₄).Concentration of the organics provided a sticky yellow solid which waspurified by chromatography on silica gel (3.5 g, 70-230 mesh) elutingwith hexane (10 mL), hexane:ether (4:1(10 mL), 3:1(10 mL), 2:1(10 mL),1:1(10 mL)), ether (30 mL), hexane:acetone (1:1 (30 mL)). One of theisomers was collected in the ether fractions.

MS (ESI) m/e 966 M)⁻

EXAMPLE 2 42 (1-tetrazolyl)-rapamycin (More Polar Isomer)

Collection of the slower moving band from the chromatography columnusing the hexane:acetone (1:1) mobile phase in Example 1B provided thedesignated compound.

MS (ESI) m/e 966 (M)⁻.

Mixed lymphocyte Reaction Assay of Rapamycin Analogs

Pharmacokinetics of Rap Analogs

The immunosuppressant activity of the compounds of embodiments of theinvention were compared to rapamycin and two rapamycin analogs:40-epi-N-[2′-pyridone]-rapamycin and 40-epi-N-[4′-pyridone]-rapamycin,both disclosed in (Or et al., 1996). The activity was determined usingthe human mixed lymphocyte reaction MLR) assay described (Kino et al.,1987). The results of the assay demonstrate that the compounds of theinvention are effective immunomodulators at nanomolar concentrations, asshown in Table 1.

TABLE 1 Human MLR Example IC₅₀ ± S.E.M. (nM) Rapamycin 0.91 ± 0.362-pyridone 12.39 ± 5.3  4-pyridone 0.43 ± 0.20 Example 1 1.70 ± 0.48Example 2 0.66 ± 0.19

The pharmacokinetic behaviors of Example 1 and Example 2 werecharacterized following a single 2.5 mg/kg intravenous dose incynomolgus monkey (n=3 per group). Each compound was prepared as 2.5mg/mL solution in a 20% ethanol:30% propylene glycol:2% cremophor EL:48%dextrose 5% in water vehicle. The 1 mL/kg intravenous dose wasadministered as a slow bolus (˜1-2 minutes) in a saphenous vein of themonkeys. Blood samples were obtained from a femoral artery or vein ofeach animal prior to dosing and 0.1 (IV only), 0.25, 0.5, 1, 1.5, 2, 4,6, 9, 12, 24, and 30 hours after dosing. The EDTA preserved samples werethoroughly mixed and extracted for subsequent analysis.

An aliquot of blood (1.0 mL) was hemolyzed with 20% methanol in water(0.5 ml) including an internal standard. The hemolyzed samples wereextracted with a mixture of ethyl acetate and hexane (1:1 (v/v), 6.0mL). The organic layer was evaporated to dryness with a stream ofnitrogen at room temperature. Samples were reconstituted inmethanol:water (1:1, 150 μL). The title compounds (50 μL injection) wereseparated from contaminants using reverse phase HPLC with UV detection.Samples were kept cool (4° C.) through the run. All samples from eachstudy were analyzed as a single batch on the HPLC.

Area under the curve (AUC) measurements of Example 1, Example 2 and theinternal standard were determined using the Sciex MacQuan™ software.Calibration curves were derived from peak area ratio parent drμg/internal standard) of the spiked blood standards using least squareslinear regression of the ratio versus the theoretical concentration. Themethods were linear for both compounds over the range of the standardcurve (correlation>0.99) with an estimated quantitation limit of 0.1ng/mL. The maximum blood concentration (C_(max)) and the time to reachthe maximum blood concentration (T_(max)) were read directly from theobserved blood concentration-time data. The blood concentration datawere submitted to multi-exponential curve fitting using CSTRIP to obtainestimates of pharmacokinetic parameters. The estimated parameters werefurther defined using NONLIN84. The area under the bloodconcentration-time curve from 0 to t hours (last measurable bloodconcentration time point) after dosing (AUC_(0-t)) was calculated usingthe linear trapeziodal rule for the blood-time profiles. The residualarea extrapolated to infinity, determined as the final measured bloodconcentration (C_(t)) divided by the terminal elimination rate constant(β), and added to AUC_(0-t) to produce the total area under the curve(AUC_(0-t)).

As shown in FIG. 1 and Table 2, both Example 1 and Example 2 had asurprisingly substantially shorter terminal elimination half-life(t_(1/2)) when compared to rapamycin. Thus, only the compounds of theinvention provide both sufficient efficacy (Table 1) and a shorterterminal half-life (Table 2).

TABLE 2 Compound AUC (ng · hr/mL) t_(1/2) (hours) Rapamycin 6.87 16.72-pyridone 2.55 2.8 4-pyridone 5.59 13.3 Example 1 2.35 5.0 Example 22.38 6.9

Paclitaxel and a Second Drug Co-administration Using a Stent

When paclitaxel is co-administered with a second drug, including arapamycin, analog or derivative, using a stent implanted in a vessel,the ratio, r, of paclitaxel:second drug by weight is such that theactivity of one drug does not attenuate the activity of the other (i.e.,interfere), and the overall effect of the co-administration is additive,and sometimes synergistic. Useful ratios of paclitaxel:second drug aregreater than approximately 7:10, or approximately 7:10≧r≧0.01:10, orapproximately 7:10≧r≧0.1:10, and or approximately r=1:10.

When applied on an implantable medical device, including a stent forblood vessel implantation, typical dosage of a therapeutic substance is0.01 μg/mm to 100 μg/mm. Typically, a practical maximum is dictated bythe polymers, the drug, and the methods of making the device. Whileother dosages are effective and useful, when paclitaxel a second drugare applied to the stent, typical dosages are 0.01 μg/mm to 20 μg/mm, or0.1 μg/mm to 15 μg/mm, and or 1 μg/mm to 10 μg/mm. However, any dosingregime can be used as long as the ratio of paclitaxel:second drug iskept within approximately 7:10≧r≧0.01:10, or approximately7:10≧r≧0.1:10, and or r=1:10 and biological safety is not significantlycompromised.

Polymer Layers and Therapeutic Substances on Medical Devices

Within the scope of the invention, there is much flexibility inproviding suitable drug-loaded polymer layers. For example, withintherapeutic window parameters (generally levels between therapeuticallyeffective and toxicity) associated with the drugs of interest, ratios ofthe drugs used in combination can be varied relative to each other. Forexample, an embodiment has a 90:10 total drug:polymer ratio with wherethe ratio of drugs in the combination can be 1:1. Thus, a stentdelivering a paclitaxel/second drug combination can include 10 μg/mmpaclitaxel and 10 μg/mm second drug in a PC polymer layer with a 5 μg/mmPC topcoat. Total drug:polymer ratio can be lower, however, e.g., 40:60or less. Upper limits on the total amount of drug will depend on severalfactors, including miscibility of the selected drugs in the selectedpolymer, the stability of the drug/polymer mixture, e.g., compatibilitywith sterilization, and the physical properties of the mixture, e.g.,flowability/processability, elasticity, brittleness, viscosity (does notweb or bridge between stent struts), coating thickness that addssubstantially to the stent profile or causes delamination or cracking oris difficult to crimp. Embodiments of the invention include stent strutsspaced about 60-80 microns apart, suggesting an upper limit in thicknessof the drug/polymer/polymer overcoat is about 30 microns; however, anystent size, strut size and spatial spacing, and/or stent constructioncan be utilized for drug delivery as described therein. Overcoatthickness (if an overcoat is used) desirably should not excessivelyimpede release kinetics of the drugs. The overcoat can also be loadedwith one or more drugs, which can be the same or different than those inthe underlying drug-loaded polymer layer.

Generally speaking, drugs useful in combinations for the invention willnot adversely affect the desired activity of the other drug in thecombination. Thus, one drug in the proposed combination will not inhibitthe desired activity, e.g., anti-proliferative activity, of the otherdrug. Nor will either drug cause or enhance the degradation of the otherdrug. However, a drug that might otherwise appear to be unsuitablebecause, for example, it degrades during sterilization, can in fact beuseful because of an interaction by another drug. Thus, dexamethasone,which alone has been observed to degrade during EtO sterilization, canbe used successfully in combination with zotarolimus, due to thehydrophobicity of zotarolimus. Moreover, zotarolimus has been observedto reduce the elution rate of dexamethasone, as described in Applicants'co-pending U.S. patent application Ser. No. 10/796,423.

Testing for Neointimal Hyperplasia and Endothelialization after StentImplantation

This test was used to determine dual drug effect on neointimalhyperplasia and endothelialization. The test exploits the art-acceptedporcine coronary overstretch model (Schwartz, R. S., Restenosis and theproportional neointimal response to coronary artery injury: results in aporcine model. J Am Coll Cardiol. February 1992; 19(2):267-274) and isusually conducted for approximately 2-8 weeks. Typically, experimentalconstruct includes at least a stent control that resembles theexperimental stent in every way except for the change of a singlevariable, including therapeutic substances or polymer.

In one example, two major coronary arteries are implanted with one teststent each, and the third major coronary artery is implanted with acontrol stent in each pig. Additional controls were pigs implanted withthree control stents, one each in a major coronary artery. Stents shouldbe the same dimensions, or as close as possible.

Stents are implanted using standard techniques. At the conclusion of thestudy, animals are euthanized, and the hearts are removed, washed andfixed using standard histological preservation techniques (includingformalin, formaldehyde, etc.). Stented vessels are excised, theninfiltrated and embedded in a suitable medium for sectioning, includingmethylmethacrylate (MMA), paraffin, or cryomedia. All blocks containingstented vessels are sectioned so that informative sections are obtained;for example, three, in-stent sections and two control sections. Serialthin sections (approximately 5 μm) are usually taken at each level andstained to visualize the cells and tissues (e.g., hematoxylin and eosin(HE) and Masson's Verhoeff Elastin (MVE)). Sections are evaluated andscored using an image analysis system or other art accepted methods ofmorphological data collection and quantification. The data are scoredfor neointimal area, neointimal thickness, and percent-area stenosis.

EXAMPLE 3

The purpose of this example was to determine the effects of a rapamycinanalog on neointimal formation in porcine coronary arteries containingstents. This example illustrates that the rapamycin analog zotarolimus,when compounded and delivered from the Biocompatibles BiodiviYsio PCCoronary stent favorably affects neointimal hyperplasia and lumen sizein porcine coronary arteries. This finding suggests that delivery ofzotarolimus from a medical device may be of substantial clinical benefitif properly applied in humans by limiting neointimal hyperplasia.

The agent zotarolimus is a rapamycin analog. The study set forth in thisexample was designed to assess the ability of the rapamycin analogzotarolimus to reduce neointimal hyperplasia in a porcine coronary stentmodel. Efficacy of zotarolimus in this model would suggest its clinicalpotential for the limitation and treatment of coronary and vascularrestenosis in stents following percutaneous revascularization. Thedomestic swine was used because this model appears to yield resultscomparable to other investigations seeking to limit neointimalhyperplasia in human subjects.

The example tested zotarolimus eluted from coronary stents placed injuvenile farm pigs, and compared these results with control stents. Thecontrol stents are polymer coated without drugs. This is important, forthe polymer itself must not stimulate neointimal hyperplasia to asubstantial degree. As the eluted drug disappears, an inflammatoryresponse to the polymer could conceivably result in a late “catch-upphenomenon” where the restenosis process is not stopped, but insteadslowed. This phenomenon would result in restenosis at late dates inhuman subjects.

Stents were implanted in two blood vessels in each pig. Pigs used inthis model were generally 2-4 months old and weighed 30-40 Kg. Twocoronary stents were thus implanted in each pig by visually assessing anormal stent:artery ratio of 1.1-1.2.

Beginning on the day of the procedure, pigs were given oral aspirin (325mg daily) and continued for the remainder of their course. Generalanesthesia was achieved by means of intramuscular injection followed byintravenous ketamine (30 mg/kg) and xylazine (3 mg/kg). Additionalmedication at the time of induction included atropine (1 mg) andflocillin (1 g) administered intramuscularly. During the stentingprocedure, an intraarterial bolus of 10,000 units of heparin wasadministered.

Arterial access was obtained by cutdown on the right external carotidand placement of an 8F sheath. After the procedure, the animals weremaintained on a normal diet without cholesterol or other specialsupplementation.

The BiodivYsio stent was used with nominal vessel target size of 3.0 mm.See FIG. 2. Two coronary arteries per pig were assigned at random todeployment of the stents. The stent was either a drug eluting stentpolymer plus drug stent) or a stent coated with a polymer only polymeronly stent). The stents were delivered by means of standard guidecatheters and wires. The stent balloons were inflated to appropriatesizes for less than 30 seconds.

Each pig had one polymer only stent and one polymer plus drug stentplaced in separate coronary arteries, so that each pig would have onestent for drug and one for control.

A sample size of 20 pigs total was chosen to detect a projecteddifference in neointimal thickness of 0.12 mm with a standard deviationof 0.15 mm, at a power of 0.95 and beta 0.02.

Animals were euthanized at 28 days for histopathologic examination andquantification. Following removal of the heart from the perfusion pumpsystem, the left atrial appendage was removed for access to the proximalcoronary arteries. Coronary arterial segments with injuries weredissected free of the epicardium. Segments containing lesions wasisolated, thereby allowing sufficient tissue to contain uninvolved bloodvessel at either end. The foregoing segments, each roughly 2.5 cm inlength, were embedded and processed by means of standard plasticembedding techniques. The tissues were subsequently processed andstained with hematoxylin-eosin and elastic-van Gieson techniques.

Low and high power light microscopy were used to make lengthmeasurements in the plane of microscopic view by means of a calibratedreticle and a digital microscopy system connected to a computeremploying calibrated analysis software.

The severity of vessel injury and the neointimal response were measuredby calibrated digital microscopy. The importance of the integrity of theinternal elastic lamina is well-known to those skilled in the art. Ahistopathologic injury score in stented blood vessels has been validatedas being closely related to neointimal thickness. This score is relatedto depth of injury and is as follows:

Score Description of Injury 0 Internal elastic lamina intact;endothelium typically denuded, media compressed but not lacerated. 1Internal elastic lamina lacerated; media typically compressed but notlacerated. 2 Internal elastic lacerated; media visibly lacerated;external elastic lamina intact but compressed. 3 External elastic laminalacerated; typically large lacerations of media extending through theexternal elastic lamina; coil wires sometimes residing in adventitia.

This quantitative measurement of injury was assessed for all stentstruts of each stent section. The calibrated digital image was also usedto measure at each stent struts site the neointimal thickness. Lumenarea, area contained with the internal elastic lamina, and area withinthe external elastic lamina were also measured.

The mid-stent segment was used for measurement, analysis, andcomparison. Data were also recorded (and included in the data section ofthis report) for proximal and distal segments.

Paired t-testing was performed to compare variables across the polymeronly stents (control group) and polymer plus drug stents (treatmentgroup). No animal died in this study before scheduled timepoints.

Table 3 shows the pigs and arteries used. In Table 3, LCX means thecircumflex branch of the left coronary artery, LAD means the leftanterior descending coronary artery, and RCA means the right coronaryartery.

TABLE 3 Pigs and Vessels Used 1 2000-G-693 RCA - Control 2000-G-693LCX - Test 2 2000-G-698 RCA - Test 2000-G-698 LAD - Control 3 2000-G-702RCA - Test 2000-G-702 LAD - Control 4 2000-G-709 RCA - Control2000-G-709 LAD - Test 5 2000-G-306 RCA - Control 2000-G-306 LAD - Test2000-G-306 *LCX - Test 6 2000-G-672 RCA - Test 2000-G-672 LAD - Control7 2000-G-712 RCA - Control 2000-G-712 LCX - Test 8 2000-G-735 RCA -Control 2000-G-735 LAD - Test 9 2000-G-736 RCA - Control 2000-G-736LCX - Test 10 2000-G-740 RCA - Test 11 2000-G-742 LAD - Test 2000-G-742OM (LCX) - Control 12 2000-G-744 RCA - Test 2000-G-744 LAD - Control 132000-G-748 RCA - Test 2000-G-748 LAD - Control 14 2000-G-749 RCA -Control 2000-G-749 LCX - Test 15 2000-G-753 RCA - Control 2000-G-753LAD - Test 16 2000-G-754 RCA - Test 2000-G-754 LCX - Control 172000-G-755 RCA - Control 2000-G-755 LAD - Test 18 2000-G-756 RCA - Test2000-G-756 LAD - Control 19 2000-G-757 LAD - Control 2000-G-757 LCX -Test 20 2000-G-760 LAD - Test 2000-G-760 LCX - Control

Table 4 shows the summary results for all data for mean injury andneointimal thickness for each stent, including proximal, mid, and distalsegments. Table 4 also shows lumen size, percent stenosis, and arterysize as measured by the internal elastic laminae (IEL) and externalelastic laminae EEL).

TABLE 4 Summary: All Measures (Distal, Mid, Proximal) ID prox ref distref lumen IEL EEL mean injury % stenosis Neointimal area NIT ControlDistal Mean 4.46 3.96 4.88 7.66 9.00 0.22 36.10 2.79 0.41 SD 1.20 1.161.30 1.15 1.10 0.26 15.41 1.29 0.17 Control Mid Mean 4.46 3.96 4.94 7.719.08 0.08 36.23 2.77 0.38 SD 1.20 1.16 1.44 1.07 1.15 0.14 14.93 1.200.16 Control Proximal Mean 4.46 3.96 5.11 7.89 9.30 0.15 35.35 2.78 0.38SD 1.20 1.16 1.38 1.33 1.42 0.22 11.94 1.04 0.12 Test Distal Mean 4.263.41 6.04 7.70 9.01 0.26 22.35 1.66 0.25 SD 1.26 0.96 1.55 1.49 1.470.43 8.58 0.58 0.06 Test Mid Mean 4.26 3.41 6.35 7.75 8.98 0.04 18.711.41 0.22 SD 1.26 0.96 1.29 1.18 1.31 0.07 5.68 0.33 0.05 Test ProximalMean 2.56 2.15 3.31 4.06 4.66 0.19 16.79 1.29 0.18 SD 1.66 1.37 2.393.48 4.15 0.13 9.97 0.80 0.12

There was no statistically significant difference for neointimal area orthickness across proximal, mid, or distal segments within the test grouppolymer plus drug stents) or control groups (polymer only stents). Thisobservation is quite consistent with prior studies, and thus allows useof only the mid segment for statistical comparison of test devices(polymer plus drug stents) vs. control devices polymer only stents).

Table 5 shows the statistical t-test comparisons across test groups andcontrol groups. There was a statistically significant difference inneointimal thickness, neointimal area, lumen size, and percent lumenstenosis, the drug eluting stent being clearly favored. Conversely,there were no statistically significant differences between the testgroup (polymer plus drug stents) and the control group (polymer onlystents) for mean injury score, external elastic laminae, or internalelastic laminae areas.

TABLE 5 Statistical Comparison of Test vs. Control Parameters:Mid-Section Data (t-test Statistics) Parameter Difference t-test DF StdError Lower 95% Upper 95% p Lumen −1.17 −2.28 38 0.52 −2.21 −0.13 0.029IEL 0.03 0.088 38 0.36 −0.71 0.78 0.93 EEL 0.2 0.499 38 0.39 −0.599 0.990.62 NI Thickness 0.18 5.153 38 0.034 0.106 0.244 <.0001 NI Area 1.213.62 38 0.33 0.53 1.88 0.0008 Mean Injury 0.038 1.137 38 0.033 −0.020.106 0.26 % Stenosis 14.54 2.97 38 4.9 4.61 24.47 0.005

The reference arteries proximal and distal to the stented segments wereobserved, and quantitated. These vessels appeared normal in all cases,uninjured in both the control group (polymer only stents) and the testgroup (polymer plus drug stents). See FIGS. 3A and 3B. The data belowshow there were no statistically significant differences in size betweenthe stents in the control group and the stents in the test group.

Proximal Reference Distal Reference Diameter (mm) Diameter (mm) Control4.46 ± 1.20 3.96 ± 1.16 (mean ± SD) Test 4.26 ± 1.26 3.41 ± 0.96 (mean ±SD)

The data demonstrates that statistically significant differences existfor morphorometric measures of efficacy favoring the stent that eluteszotarolimus. The stent of this invention results in lower neointimalarea, lower neointimal thickness, and greater lumen area. There were nosignificant differences within the test group (polymer plus drug stents)and the control group (polymer only stents) for inflammation or injuryparameters. There were no significant differences in artery sizes(including the stent) for the control group compared to the test group.These latter findings suggest no significant difference in the arterialremodeling characteristics of the polymeric coating containing the drug.

At most, mild inflammation was found on both the polymer plus drug stentand the polymer only stent. This finding suggests that the polymerexhibits satisfactory biocompatibility, even without drug loading. Otherstudies show that when drug has completely gone from the polymer, thepolymer itself creates enough inflammation to cause neointima. Thisobservation may be responsible for the late catch-up phenomenon ofclinical late restenosis. Because the polymer in this example did notcause inflammation in the coronary arteries, late problems related tothe polymer after the drug is exhausted are unlikely.

In conclusion, a stent eluting the compound zotarolimus from a polymershowed a reduction in neointimal hyperplasia in the porcine model whenplaced in a coronary artery.

EXAMPLE 4

The purpose of this example is to determine the rate of release of thezotarolimus drug from 316L Electropolished Stainless Steel Couponscoated with a biocompatible polymer containing phosphorylcholine sidegroups.

Rubber septa from lids from HPLC vials were removed from the vials andplaced into glass vials so that the “Teflon” side faced up. These septaserved as supports for the test samples. The test samples were 316Lstainless steel coupons that had been previously coated with abiocompatible polymer containing phosphorylcholine side groups PCpolymer). Coronary stents are commonly made of 316L stainless steel andcan be coated with the PC polymer to provide a depot site for loadingdrugs. The coated coupons, which serve to simulate stents, were placedonto the septa. By using a glass Hamilton Syringe, a solution ofzotarolimus and ethanol (10 μl) was applied to the surface of eachcoupon. The solution contained zotarolimus (30.6 mg) dissolved in 100%ethanol (3.0 ml). The syringe was cleaned with ethanol between eachapplication. The cap to the glass vial was placed on the vial loosely,thereby assuring proper ventilation. The coupon was allowed to dry for aminimum of 1.5 hours. Twelve (12) coupons were loaded in this way—sixbeing used to determine the average amount of drug loaded onto thedevice and six being used to measure the time needed to release the drugfrom the devices.

To determine the total amount of zotarolimus loaded onto a coupon, acoupon was removed from the vial and placed into 50/50acetonitrile/0.04M phosphate buffer (pH 6.0, 5.0 ml). The coupon wasplaced onto a 5210 Branson sonicator for one hour. The coupon was thenremoved from the solution, and the solution was assayed by HPLC.

The time release studies were performed by immersing and removing theindividual coupons from fresh aliquots (10.0 ml) of 0.01 M phosphatebuffer at a pH of 6.0 at each of the following time intervals—5, 15, 30and 60 minutes. For the remaining time points of 120, 180, 240, 300, 360minutes, volumes of 5.0 ml of buffer were used. To facilitate mixingduring the drug release phase, the samples were placed onto an Eberbachshaker set at low speed. All solution aliquots were assayed by HPLCafter the testing of the last sample was completed.

The HPLC analysis was performed with a Hewlett Packard series 4400instrument having the following settings:

Injection Volume = 100 μl Acquisition Time = 40 minutes Flow Rate = 1.0ml/min Column Temperature = 40° C. Wavelength = 278 nm Mobile Phase =65% Acetonitrile/35% H₂O Column = YMC ODS-A S5 μm, 4.6 × 250 mm Part No.A12052546WT

The results from the above experiment showed the following release data(Table 6):

TABLE 6 Time (min.) Percent Release Standard Deviation 0.00 0.00 0.005.00 1.87 1.12 15.00 2.97 1.47 30.00 3.24 1.28 60.00 3.29 1.29 120.003.92 1.28 180.00 4.36 1.33 240.00 4.37 1.35 300.00 6.34 2.07 360.00 7.881.01

EXAMPLE 5

The purpose of this example was to determine the loading and release ofzotarolimus from 15 mm BiodivYsio drug delivery stents.

To load the stents with drug, a solution of zotarolimus in ethanol at aconcentration of 50 mg/ml was prepared and dispensed into twelve vials.Twelve individual polymer-coated stents were placed on fixtures designedto hold the stent in a vertical position and the stents were immersedvertically in the drug solution for five minutes. The stents andfixtures were removed from the vials and excess drug solution wasblotted away by contacting the stents with an absorbent material. Thestents were then allowed to dry in air for 30 minutes in an invertedvertical position.

The stents were removed from the fixtures, and each stent was placedinto 50/50 acetonitrile/phosphate buffer pH 5.1, 2.0 ml) and sonicatedfor one hour. The stents were removed from the solution and solutionswere assayed for concentration of drug, which allowed calculation of theamount of drug originally on the stents. This method was independentlyshown to remove at least 95% of the drug from the stent coating. Onaverage, the stents included 120±9 micrograms of drug.

The drug-loaded stents were placed on the fixtures and placed into 0.01M phosphate buffer (pH=6.0, 1.9 ml) in individual vials. These sampleswere placed onto a Eberbach shaker set at low speed to provideback-and-forth agitation. To avoid approaching drug saturation in thebuffer, the stents were transferred periodically to fresh buffer vialsat the following points: 15, 30, 45, 60, 120, 135, 150, 165, 180, 240,390 minutes. The dissolution buffer vials were assayed by HPLC for thedrug concentration at the end of the drug release period studied. Thedata, represented as % cumulative release of the drug as a function oftime, is shown in tabular form below (Table 7):

TABLE 7 Time (min) % Cumulative Release of Drug 15 0.3 30 1.1 45 2.1 603.2 120 4.3 135 5.9 150 6.3 165 6.8 180 7.4 240 10.8 390 13.2

EXAMPLE 6

Zotarolimus, a tetrazole analog of rapamycin, has been shown to possessanti-restenosis activity in swine coronary stent-induced injury andRobinson, K A, Dube, H. M. Efficacy Evaluation of zotarolimus LoadedCoronary Stents in Yucatan Miniswine—Preclinical Laboratory Study, Sep.20, 2001 and Schwartz, R. S. Efficacy Evaluation of a Rapamycin Analog(A-179578). Delivered from the Biocompatibles BiodivYsio PC CoronaryStents in Porcine Coronary Arteries, Technical Report, Mayo Clinic andFoundation, Rochester, Minn.) rat balloon angioplasty (Gregory, C.Summary of Study Evaluating Effects of zotarolimus in a Rat Model ofVascular Injury) models. The objective of this example was to assess thesafety and pharmacokinetics (PK) of escalating single intravenous (IV)doses of zotarolimus in healthy males.

In the present, first-time-in-man study, the safety and pharmacokineticsof zotarolimus were investigated following intravenous bolusadministration of zotarolimus over a 100 to 900 μg dose range. Theintravenous bolus dose administration would mimic the most rapidunexpected release of zotarolimus from drug-coated stents in vivo.

This was a Phase 1, single escalating dose, double-blind, randomized,placebo-controlled, single-center study. Sixty (60) adult healthy maleswere divided into 5 IV dose groups of 100, 300, 500, 700, and 900 μg.Demographic information for the subjects is summarized in Table 8.

TABLE 8 Demographic Summary for All Subjects Mean ± SD (N = 60) Min-MaxAge (years) 32.6 ± 7.1 19-44 Weight (kg)  80.0 ± 10.6  62-104 Height(cm) 180.5 ± 7.2  160-195 Race 60 Caucasians (100%)  

Subjects were randomly assigned to receive a single intravenous dose ofzotarolimus or a matching intravenous placebo under fasting conditions,as shown in the dosing scheme shown in Table 9.

TABLE 9 Treatment Group Double-blind Treatment Number of Subjects I 100μg zotarolimus/Placebo 8/4 II 300 μg zotarolimus/Placebo 8/4 III 500 μgzotarolimus/Placebo 8/4 IV 700 μg zotarolimus/Placebo 8/4 V 900 μgzotarolimus/Placebo 8/4

Higher doses were administered after evaluating the safety data from thepreceding lower dose groups. The treatment groups were separated by atleast 7 days. For safety reasons, each treatment group was divided intotwo cohorts of six subjects and the doses of the two cohorts of a groupwere separated by at least 1 day.

Doses were administered as IV bolus over 3 minutes, with 8 subjects.Four subjects received zotarolimus and 4 subjects received placebo ineach dose group. Blood concentrations of zotarolimus were sampled for168 hours and measured using LC-MS/MS with a LOQ of 0.20 ng/mL

Seven (7)-mL blood samples were collected by venipuncture into evacuatedcollection tubes containing edetic acid (EDTA) prior to dosing (0 hour)and at 0.083 (5 min), 0.25, 0.5, 1, 2, 4, 8, 12, 16, 24, 36, 48, 72, 96,120, 144, and 168 hours after dosing on Study Day 1.

Blood concentrations of zotarolimus were determined using a validatedliquid/liquid extraction HPLC tandem mass spectrometric method LC-MS/MS)(Ji et al., 2004). The lower limit of quantification of zotarolimus was0.20 ng/mL using 0.3 mL blood sample. All calibration curves hadcoefficient of determination (r²) values greater than or equal to0.9923.

Safety was evaluated based on adverse event, physical examination, vitalsigns, ECG, injection site and laboratory tests assessments.

Pharmacokinetic parameter values of zotarolimus were estimated usingnoncompartmental methods. These parameters included: concentration at5-minutes zotarolimus post-dose (C₅), dose-normalized C₅, eliminationrate constant (β), half-life (t_(1/2)), the area under the bloodconcentration vs. time curve from time 0 to time of the last measurableconcentration (AUC_(0-last)), dose-normalized AUC_(0-last), the areaunder the blood concentration vs. time curve extrapolated to infinitetime (AUC_(0-inf)), dose-normalized AUC_(0-inf), total clearance (CL),and volume of distribution (Vd_(β)).

Mean blood concentration-time plots, following intravenous doses ofzotarolimus are presented in FIGS. 4 and 5 on linear scale andlog-linear scale, respectively.

Mean±SD pharmacokinetic parameters of zotarolimus after administrationof each of the two regimens are shown in Table 10.

TABLE 10 Mean ± SD Pharmacokinetic Parameters of zotarolimus Dose ofzotarolimus Pharmacokinetic Parameters 100 μg (N = 8) 300 μg (N = 8) 500μg (N = 8) 700 μg (N = 8) 900 μg (N = 8) C₅ (ng/mL) 13.48 ± 2.87  36.71± 9.82*  56.50 ± 27.54* 88.73 ± 5.00  110.78 ± 15.91* C₅/Dose (ng/mL/μg)0.13 ± 0.03 0.12 ± 0.03 0.11 ± 0.05 0.13 ± 0.01 0.12 ± 0.02 AUC_(0-last)(ng · h/mL) 24.57 ± 5.89  77.79 ± 13.70 146.04 ± 32.39  207.92 ± 19.44 240.80 ± 19.19  AUC_(0-last)/Dose (ng · h/mL/μg) 0.25 ± 0.06 0.26 ± 0.050.29 ± 0.06 0.30 ± 0.03 0.27 ± 0.02 AUC_(0-inf) (ng · h/mL) 35.28 ±6.15  91.17 ± 14.68 162.44 ± 29.58  221.77 ± 19.60  254.47 ± 17.60 AUC_(0-inf)/Dose (ng · h/mL/μg)^(#) 0.35 ± 0.06 0.30 ± 0.05 0.32 ± 0.060.32 ± 0.03 0.28 ± 0.02 β (1/h)^(#) 0.027 ± 0.006 0.019 ± 0.002 0.017 ±0.003 0.020 ± 0.001 0.018 ± 0.002 t_(1/2) (h)^($) 26.0 ± 6.0  35.9 ±4.6  40.2 ± 7.8  35.0 ± 2.4  39.0 ± 3.9  CL (L/h) 2.90 ± 0.44 3.36 ±0.50 3.17 ± 0.58 3.18 ± 0.28 3.55 ± 0.24 Vd_(β) (L)^(#) 113 ± 23  175 ±23  190 ± 49  161 ± 15  202 ± 29  ^($)Harmonic mean ± pseudo-standarddeviation; evaluations of t_(1/2) were based on statistical tests for β;A > 10% sampling time deviation occurred for the 5-minutes sample forSubjects 201, 304, and 512; C₅ concentrations for these subjects werenot calculated. (N = 7); ^(#)Statistically significant monotonic trendwith dose

To investigate the questions of dose proportionality and linearpharmacokinetics, an analysis of covariance (ANCOVA) was performed.Subjects were classified by dose level, and body weight was a covariate.The variables analyzed included β, Vd_(β), dose-normalized C₅, andlogarithms of dose-normalized AUC_(0-last) and dose-normalizedAUC_(0-inf). The primary test of the hypothesis of invariance with dosewas a test on dose-level effects with good power for a monotonicfunction of dose. In addition, the highest and lowest dose levels werecompared within the framework of the ANCOVA

FIG. 6 depicts the dose proportionality of zotarolimus C_(max),AUC_(0-last), and AUC_(0-inf). As can be seen in this figure, nostatistically significant monotonic trend was observed with dosenormalized C_(max), and AUC_(0-last) suggesting a dose proportionalincrease in these parameters. A statistically significant monotonictrend with dose was observed for the dose-normalized AUC_(0-inf) ofzotarolimus (p=0.0152). However, a pairwise comparison ofdose-normalized AUC_(0-inf) across all groups showed that only 100 μgdose-normalized AUC_(0-inf) was statistically significant different fromthat of 900 μg and 300 μg (p=0.0032 and p=0.0316, respectively). Astatistically significant monotonic trend was also observed with β. Thisdeparture could be due to slight overestimation of β with the 100 μgdose group. The mean zotarolimus C₅ (concentration at 5 minutes) andAUC_(0-inf) increased proportionally with dose, as shown in Table 11.

TABLE 11 Pharmacokinetic Dose (μg) (N = 8) Parameters 100 300 500 700900 C₅ (ng/mL) 13.48 ± 2.87 36.71 ± 9.82  56.50 ± 27.54 88.73 ± 5.00110.78 ± 15.91 AUC_(0-inf) (ng · h/mL) 35.28 ± 6.15 91.17 ± 14.68 162.44± 9.58  221.77 ± 19.60 254.47 ± 17.60 CL (L/h)  2.90 ± 0.44 3.36 ± 0.503.17 ± 0.58  3.18 ± 0.28  3.55 ± 0.24

The mean half-life ranged between 26.0-40.2 h over the studied doses andwas not significantly different over the 300-900 μg dose range.Zotarolimus was well tolerated at all doses and no clinicallysignificant physical examination results, vital signs or laboratorymeasurements were observed.

Safety

The most common treatment-emergent adverse events (reported by two ormore subjects in any one treatment group) associated with zotarolimuswere injection site reaction and pain.

The majority of the adverse events was mild in severity and resolvedspontaneously.

There were no serious adverse events reported in this study.

There were no clinically significant changes in physical examinationfindings, vital signs, clinical laboratory or ECG parameters during thestudy.

Conclusion

The pharmacokinetics of IV zotarolimus are dose-proportional over the100-900 μg dose range with respect to C₅ and AUC_(0-inf). Overall, thepharmacokinetics of zotarolimus were essentially linear across the 100μg to 900 μg dose range as illustrated by the dose proportionalincreases in C₅, AUC_(0-last), and AUC_(0-inf). Single IV bolus doses upto 900 μg were administered without safety concerns.

Mean elimination half-life of zotarolimus ranged from 26.0 to 40.2 hoursover the studied dose range. The mean clearance and volume ofdistribution ranged from 2.90 to 3.55 L/h and 113 to 202 L,respectively. The observed departure from linear kinetics for β and, toa significant extent, for Vd_(β) was due to an overestimation of β forthe 100 μg dose group.

Zotarolimus in single doses of 100 to 900 μg were generally welltolerated by the subjects.

EXAMPLE 7

The present study was designed to evaluate the pharmacokinetics ofzotarolimus following multiple dosing and to assess its safety whilemaximizing systemic exposure of healthy subjects. The primary goal wasto achieve a total exposure of zotarolimus significantly above theanticipated levels of the drug eluted from coated stents. The studyinvestigated pharmacokinetics and safety of zotarolimus in a Phase 1,multiple dose-escalation study following multiple intravenous infusionsof 200, 400 and 800 μg doses, every day for fourteen consecutive days inhealthy subjects.

Methods

Phase 1, multiple-escalating dose, double-blind, placebo-controlled,randomized study. Seventy-two subjects equally divided in 3 once-daily(QD) regimens (200, 400 or 800 μg QD with 16 active and 8 placebo perregimen) were administered a 60-minute QD IV infusion of zotarolimus for14 consecutive days. Blood samples were collected over 24 hoursfollowing the first dose, before dosing on days 10, 11, 12, 13, and for168 hours following Day 14 dose. Urine samples were collected over 24hours on days 1, 14, 16, 18 and 20. Blood and urine zotarolimusconcentrations were determined using a validated LC/MS/MS method.Pharmacokinetic parameters were determined by compartmental analysis.All Day-AUC_(0-∞) (area under blood concentration-time curve from time 0to infinity including all 14 doses) was calculated. Dose andtime-linearity and achievement of steady-state were evaluated. Fractionof drug eliminated in urine was determined.

Seventy-two (72) male and female subjects in general good health wereenrolled in this study. Demographic information is summarized in Table12.

TABLE 12 Demographic Summary for All Randomized Group I, Group II andGroup III Subjects Mean ± SD (N = 72) Min-Max Age (years) 36.9 ± 7.819-59 Weight (kg) 78.0 ± 8.2 61-97 Height (cm) 178.5 ± 6.3  163-193 Sex70 Males (97%), 2 Females (3%) Race 71 White (99%), 1 Black (1%)

Subjects were randomized at two different sites to three groups (GroupsI, II and III) as shown in Table 13. Within each group, subjects wereequally divided at the two study sites with each site enrolling 12subjects (zotarolimus, eight subjects; placebo four subjects). Thedosing scheme within each dose group is presented below:

TABLE 13 Dosing Scheme Number of Group Subjects Double-Blind IVTreatment I  16⁺ 200 μg zotarolimus over 60 min QD for 14 days  8Placebo over 60 min QD for 14 days II 16 400 μg zotarolimus over 60 minQD for 14 days  8 Placebo over 60 min QD for 14 days III 16 800 μgzotarolimus over 60 min QD for 14 days  8 Placebo over 60 min QD for 14days ⁺Subject 2112 prematurely discontinued the study; subject withdrewconsent on Study Day 19.

Subjects received, under fasting conditions, a single 60-minute daily(QD) intravenous infusion of 200, 400, or 800 μg of zotarolimus or amatching intravenous infusion of placebo for Groups I, II and III,respectively on Study Days 1 through 14. The drug was administered via asyringe pump connected to a y-site device, which also infused 125-150 mLof 5% aqueous dextrose solution D5W) over 60 minutes. The groups weredosed sequentially with at least 7 days separating the last dose of theprevious group and the first dose of the next group during which timesafety data from the previous group was analyzed. Dose escalation wasdependent on the safety analysis of the lower dose group.

Five (5)-mL blood samples were collected in potassium EDTA containingtubes to evaluate zotarolimus concentrations prior to dosing (0 hour),and at 0.25, 0.5, 1.0, 1 hour 5 min, 1.25, 1.5, 2, 3, 4, 8, 12, 18 and24 hours after starting infusion on Study Days 1 and 14. Additionalsamples were collected at 36, 48, 72, 96, 120, 144 and 168 hours afterstarting infusion on Study Day 14 and before dosing on Days 10, 11, 12and 13. Urine was collected in containers without preservatives over thefollowing intervals: 0 to 6, 6 to 12, 12 to 18 and 18 to 24 hours afterstarting the infusion on Study Days 1, 14, 16, 18 and 20.

Blood and urine concentrations of zotarolimus were determined using avalidated liquid/liquid extraction HPLC tandem mass spectrometric method(LC-MS/MS). The lower limit of quantification of zotarolimus was 0.20ng/mL using 0.3 mL blood sample and 0.50 ng/mL using 0.3 mL urinesample.

Safety was evaluated based on adverse event, physical examination, vitalsigns, ECG, injection site and laboratory tests assessments

Results

Zotarolimus blood concentration-time data for all subjects weredescribed by a three compartment open model with first orderelimination. Over the studied regimens, the range of mean compartmentalpharmacokinetic parameters were: CL 4.0-4.6 L/h; V₁ 11.3-13.1 L; V_(SS)92.5-118.0 L, and terminal elimination t_(1/2) 24.7-31.0 h. Zotarolimuspharmacokinetics were consistent with dose linearity over the studiedregimens, on days 1 and 14. The pharmacokinetic model simultaneously fitdata for days 1 and 14, indicating time-linear pharmacokinetics. AllDay-AUC_(0-∞) for the studied regimens ranged from 677-2395 ng·hr/mL. Onaverage, 0.1% of zotarolimus dose was recovered in the urine within a24-hour period post-dose.

Pharmacokinetic and Statistical Analysis

The pharmacokinetic parameter values of zotarolimus were estimated forindividual subjects using compartmental analysis. Data from the firstdose on Study Day 1, the last dose on Study Day 14 and the troughconcentrations on Study Days 10, 11, 12 and 13 were simultaneouslymodeled for each individual subject. Parameters determined were: volumeof the central compartment (V₁), terminal elimination rate constantgamma), clearance (CL), volume of distribution at steady state V_(SS)),half-life (t_(1/2)), maximum concentration (C_(max)), time of maximumconcentration (T_(max)), area under the blood concentration versus timecurve for Day 14 (AUC_(τ)) and corresponding dose normalized C_(max) andAUC_(τ). The optimal model for each individual was used to predict theindividual's concentration-time profile over a 14-day period to estimatethe chronic exposure over the study duration, i.e., C_(max) and AllDay-AUC_(0-∞) (Area under the predicted blood concentration-time profilefrom time 0 to infinity taking into account all 14 doses in the study).

To assess dose proportionality for the Study Day 14 dose an analysis ofcovariance (ANCOVA) for the logarithm of dose-normalized C_(max),dose-normalized AUC, and terminal elimination rate constant□ wasperformed. The center and the dose were factors and body weight was acovariate. To address the question of whether steady state was reached,a repeated measures analysis, with center and dose level as factors, wasperformed on the dose-normalized pre-dose concentrations of Study Days10-14.

Pharmacokinetics

Zotarolimus blood concentration-time data for all subjects weredescribed by a three compartment open model with first orderelimination. The mean blood concentrations for zotarolimus for Day 1,Day 14 and Days 1 through 14 are presented in FIG. 7. The mean±SD ofpharmacokinetic parameters of zotarolimus are presented in Table 14.

TABLE 14 Mean ± SD Compartmental Pharmacokinetic Parameters ofzotarolimus Dose Groups Pharmacokinetic 200 μg QD 400 μg QD 800 μg QDParameters (units) (N = 15) (N = 16) (N = 16) V₁ (L) 11.4 ± 1.7  11.3 ±1.0  13.1 ± 3.2  Gamma (h-1) 0.028 ± 0.005 0.022 ± 0.003 0.023 ± 0.003C_(max)* (ng/mL) 11.2 ± 1.1  21.4 ± 2.4  38.7 ± 6.3  C_(max)/ (ng/mL/μg)0.056 ± 0.006 0.053 ± 0.006 0.048 ± 0.008 Dose* AUC_(τ)* (ng · h/mL)49.0 ± 6.2  104.2 ± 19.0  179.5 ± 17.4  AUC_(τ)/ (ng · h/mL/ 0.245 ±0.031 0.260 ± 0.047 0.224 ± 0.022 Dose* μg) t_(1/2)$* (h) 24.7 ± 4.6 31.0 ± 4.6  30.0 ± 4.1  CL* (L/h) 4.2 ± 0.6 4.0 ± 0.9 4.6 ± 0.4 V_(SS)*(L) 92.5 ± 13.0 111.5 ± 21.1  118.0 ± 18.7  $Harmonic mean ±pseudo-standard deviation *Secondary predicted parameters

As no bias in the observed versus predicted diagnostic plots over thestudied regimens was observed, the ranges of the compartmentalpharmacokinetic parameters over the studied dose regimens were verynarrow and no meaningful trend over the studied dose regimens in thesecondary parameters was observed; dose linearity was inferred forzotarolimus over the studied dose regimens.

The following figure depicts the dose proportionality in zotarolimus Day14 C_(max) and AUC_(0-24h) FIGS. 8 a, 8 b and 8 c show mean zotarolimusblood concentration-time profiles for the 200, 400 and 800 μg QD dosegroups on Day 1, Day 14 and Days 1-14, respectively. For each dosegroup, the model adequately described the data on Day 1 as well as Day14 and in between as exemplified in FIG. 9 (example of mean observed andpredicted blood concentration versus time plots upon fitting 800 μg QDdose group data). The excellent fit of the observed zotarolimusconcentration-time data over Days 1 through 14 by a 3-compartment modelthat assumes linear kinetics indicates that zotarolimus exhibits timeinvariant clearance.

As shown in FIG. 9, no statistical differences were observed in thedose-normalized pre-dose concentrations of Study Days 10-14.

The median C_(max) for the 200, 400 and 800 μg QD dose groups was 11.4,22.1 and 38.9 ng/mL, respectively. The corresponding median AllDay—AUC_(0-∞) was 677, 1438, and 2395 ng·h/mL, respectively.

The fraction of the zotarolimus dose eliminated in the urine wascalculated for the 800 μg QD dose group. On average, approximately 0.1%of zotarolimus was recovered in the urine within a 24-hour period on Day1 and Day 14.

Safety

The most common treatment-emergent adverse events associated withzotarolimus were pain, headache, injection site reaction, dry skin,abdominal pain, diarrhea and rash. The majority of the adverse eventswere mild in severity and resolved spontaneously. There were no seriousadverse events reported in this study. Specifically, no subjectdisplayed any clinical or biochemical evidence of immunosuppression, QTcprolongation or clinically significant adverse events.

Conclusions

Zotarolimus pharmacokinetics were dose proportional and time invariantwhen administered intravenously for 14 consecutive days, over thestudied dose regimens.

Steady state for QD dosing of zotarolimus was reached by Day 10, the dayon which the first trough samples were measured.

Renal excretion is not a major route of elimination for zotarolimus asapproximately 0.1% of the dose was excreted as unchanged drug in theurine per day.

Zotarolimus is generally well tolerated when given in multiple doses of200, 400, and 800 μg for 14 consecutive days.

EXAMPLE 8 Anti-proliferative Activity of Zotarolimus and Paclitaxel

Experiments were performed to investigate interactions betweenzotarolimus (ABT-578) and paclitaxel when administered in combination.The effects of paclitaxel and zotarolimus on the anti-proliferativeactivity of human coronary artery smooth muscle hCaSMC) and endothelialcells hCaEC) were determined using an in vitro proliferation assay. Theproliferation and migration of vascular smooth muscle cells into thevascular neointima is a characteristic pathologic response seen inrestenotic lesions Lafont and Libby, 1998). As a result, in vitro assayswhich specifically measure the anti-proliferative activity of candidateanti-restenotic compounds on human coronary artery smooth muscle andendothelial cells predict potential anti-restenotic activity in vivo.

Compounds or combinations of compounds which attenuate growthfactor-mediated human coronary artery smooth muscle cell (hCaSMC)proliferation, as measured by the tritium incorporation assay in vitro,are candidate anti-restenotic agents. The tritium incorporation assay isan accurate and sensitive method to determine cell number andproliferation. This assay was employed to determine if agents whichdemonstrate anti-proliferative activity alone also demonstrate similaractivity in combination. Furthermore, agents which demonstrate lowerpotency anti-proliferative activity may block the activity of morepotent anti-proliferative agents when administered in combination. Theattenuation of zotarolimus's anti-proliferative activity by tacrolimusis a clear example of this effect (FIG. 10A). To determine the potentialanti-restenotic activity of combinations of zotarolimus and paclitaxel,the proliferation of hCaSMC was measured in the presence of eachcompound and in combination.

Paclitaxel interferes with microtubule de-polymerization, blocking cellprogression at the S phase (Schiff and Horwitz, 1980). Zotarolimus, likerapamycin, blocks cyclin-dependent kinase via mTOR inhibition andinhibits cell cycle progression at the G1-S phase Marx et al., 1995;Sehgal, 1998; Sehgal, 2003).

To determine if paclitaxel attenuated or augmented the activity ofzotarolimus the effect of paclitaxel and zotarolimus alone and incombination on growth-factor induced proliferation was determined. Datawere analyzed for additivity using an isobologram approach and acombination index analysis. An isobologram is a Cartesian plot of pairsof doses that, in combination, yield a specified level of effect. It isa convenient way of graphically displaying results of drug-combinationand similar studies, because paired values of experimental points thatfall below or above the line connecting the axial points indicatesynergistic and non-synergistic interactions, respectively.

Proliferation Assay Methods

³H-thymidine Uptake

Cell proliferation was monitored by following incorporation of³H-thymidine into newly synthesized DNA of cells stimulated by serum andgrowth factors. Exponentially growing hCaSMCs were seeded into 96-wellflat bottom tissue culture plates at 5,000 cells/well (10,000 cells/wellfor hCaECs). The cells were allowed to attach overnight. The growthmedium was removed the following day, and cells were washed twice withun-supplemented (basal) medium to remove traces of serum and growthfactors. Basal medium (200 μl) was added to each well and the cellsincubated in medium lacking growth factors and serum to starve andsynchronize them in the G0 state. After starvation (48 hours for hCaSMCsand 39 hours for hCaECs) in medium lacking serum and growth factors, thecells were replenished with 200 μl supplemented medium in the absence orpresence of drugs. Dimethylsulfoxide (DMSO) was maintained at a finalconcentration of 0.1% in all wells. After a 72-hour incubation period,25 μl (1 μCi/well) of ³H-thymidine (Amersham Biosciences; Piscataway,N.J.) were added to each well. The cells were incubated at 37° C. for16-18 hours, and the cells harvested onto 96-well plates containingbonded glass fiber filters using a cell harvester (Harvester 9600,TOMTEC; Hamden, Conn. The filter plates were air dried overnight andMicroScint-20 (25 μl; PerkinElmer; Wellesley; MA) was added to eachfilter well and the plates were counted using a TopCount microplatescintillation counter (PerkinElmer). Controls included medium only,starved cells and cells in complete medium. Drug activity wasestablished by determining the inhibition of ³H-thymidine incorporationinto newly synthesized DNA relative to cells grown in complete medium.

The data are presented as percent inhibition of ³H-thymidineincorporation relative to vehicle-treated controls and are given as themean±SEM of 3-4 experiments. A semi-log plot of the average values ofinhibition from each experiment versus drug concentration was generated,and the IC₅₀ (Median Inhibition Concentration (concentration thatreduces cell proliferation by 50%) for each experiment was determined byextrapolation of the 50% inhibition level relative to cells incubated incomplete medium in the absence of drugs. The final IC₅₀'s are means ofthe 3-4 experiments.

In these experiments, the x-axis represents the concentration of thedrug being varied. Each graph contains a zotarolimus- andpaclitaxel-alone curve. The set of curves in each graph was generated byadding paclitaxel at a fixed concentration to the indicatedconcentrations of zotarolimus. Each curve represents the dose-responseof zotarolimus (concentration given on the x-axis) in the presence ofthe indicated fixed concentration of paclitaxel.

Two methods were used to analyze the combined effects of zotarolimus andpaclitaxel on proliferation. Isobolograms were generated at severaleffect levels (Tallarida et al., 1989). The concentration responsecurves were fit by non-linear regression Prism, GraphPad Software; SanDiego, Calif.) to obtain EC50 and hill slope values. The concentrationeliciting a specific anti-proliferative effect was determined using afour-parameter equation (equation 1):

$\begin{matrix}{{Y = {{Bottom} + {\left( {{Top} - {Bottom}} \right)/\left( {1 + {10\hat{}\left( {\left( {{{Log}\;{EC50}} - X} \right)*{HillSlope}} \right)}} \right)}}}\mspace{79mu}{X\mspace{14mu}{is}\mspace{14mu}{the}\mspace{14mu}{logarithm}\mspace{14mu}{of}\mspace{14mu}{{concentration}.\mspace{14mu} Y}\mspace{14mu}{is}\mspace{14mu}{the}\mspace{14mu}{response}}\mspace{79mu}{{Alternately}\text{:}}\mspace{79mu}{Y = {{Bottom} + \frac{\left( {{Top} - {Bottom}} \right)}{\frac{1 + {EC}_{50}^{Hillslope}}{\lbrack X\rbrack^{Hillslope}}}}}} & \lbrack 1\rbrack\end{matrix}$Where X=log concentration of drug yielding Y response and top and bottomvalues are constrained to 100 and 0, respectively. In addition toisobolograms, the data were analyzed for synergism using the method ofChou and Talalay (Chou and Talalay, 1984) with the following exception.The regression model generated for each curve was used in place ofmedian-effect data (log-logit plot) because the non-linearfour-parameter equation more accurately models theconcentration-response curve. The median-effect plot is heavilyinfluenced by values of fractional occupancy below 0.2 and greater than0.8. The combination indices (CI) for several drug combinations yielding25%, 50%, 60% and 75% were calculated according to equation 2.(D)₁/(D _(x))₁+(D)₂/(D _(x))₂+((D)₁(D)₂)/(D _(x))₁(D _(x))₂=CI  (equation 2)

where at a specified effect level (D)₁ and (D)₂ are the concentrationsof drug 1 and drug 2 in the combination and (D_(x))₁ and (D_(x))₂ arethe concentrations of drug 1 alone and drug 2 alone. CI values reflectthe summation of effects of the combinations assuming each drug wasacting in accordance with its own potency. Equation 2 describespredicted effects for the combination of two mutually nonexclusivecompounds. If each drug contributes to the combined effect in accordanceto its own dose-dependent fractional occupancy then the CI is equalto 1. Values of CI below one are considered synergistic and valuessignificantly over one are considered sub-additive. Since therelationship between CI, and synergism, additivity or attenuation can beeffect-level dependent, CI was determined at several effect levels usingmultiple drug combinations. CI values were plotted as a function of theeffect level (or fa) at which they were calculated. CI values, similarto the isobologram analysis are effect level dependent and vary as thelevel of effect changes therefore it is important to consider effectlevel in comparing CI values. The accuracy of CI values are, in turndependent on the accuracy of the concentration values used in theircalculation. In this study an accurate method (iterative curve fittingby GraphPad software) was used to calculate drug concentrations fromeach cumulative dose-response curve at several effect levels.Dose-response curves can be fit to data which may demonstrate littledose-dependent activity. This is particularly apparent when analyzingdose-response curves generated in the presence of a high concentrationof one of the test agents. Errors in determination of drugconcentrations from the dose-response curves under these conditions mayresult in high CI values at low effect levels (fa). Therefore, CI valuesgenerated from well defined dose-response curves near or abovehalf-maximal effects (i.e., fa˜0.5) are the most accurate predictors ofthe activity of drug combinations. Under these conditions values of CIbelow one are considered supra-additive and values significantly overone are considered sub-additive. Values near one are consideredadditive.

Results

This study addressed the activity of agents on two cell types implicatedin restenosis, human coronary artery smooth muscle (hCaSMC) andendothelial cells hCaEC). The results are given in FIG. 10 and Table 15.FIG. 10 shows that tacrolimus blocks the anti-proliferative activity ofzotarolimus in smooth muscle cells in vitro (FIG. 10A). Theanti-proliferative activity of zotarolimus, paclitaxel P) andcombinations in smooth muscle cells (FIG. 10B) and endothelial cells(FIG. 10C) in vitro are also shown. FIGS. 10D-G show isobologramanalyses of combination anti-proliferative activity in smooth musclecells. The concentrations producing the specified level ofanti-proliferative activity were determined from the dose-responsecurves generated by non-linear curve fitting of the data means. FIGS.10H-K shows isobologram analyses of the anti-proliferative activity ofthe combination of zotarolimus and paclitaxel in endothelial cells. Theconcentrations of compounds producing the specified levels of activitywere determined from the mean data. FIGS. 10L-M shows a combinationindex (CI) analysis of the antiproliferative activity of combinations ofABT-578 and paclitaxel in hCaSMC and hCaEC. CI levels were determinedfrom the mean data using the method of Chou and Talalay (Chou andTalalay, 1984).

The data from each individual agent alone show that both zotarolimus andpaclitaxel dose-dependently inhibit proliferation in each cell type.FIG. 10B/C shows that the inhibition of proliferation by zotarolimus isnot blocked by paclitaxel. Increasing concentrations of both paclitaxeland zotarolimus almost completely inhibit hCaEC and hCaSMCproliferation. These data show that at low effect levels (i.e., ≦50%inhibition of proliferation) the effects of combining paclitaxel andzotarolimus are predicted by the sum of their individual activity. Thisrelationship holds at most levels of inhibition except at high levels ofinhibition. At high levels of inhibition, the anti-proliferativeactivity slightly exceeds that predicted by the activity of each agentalone. Both the isobologram and CI analyses of the hCaSMC data show thatthe combination containing paclitaxel (2.5 nM) and zotarolimusdemonstrate potential supra-additive anti-proliferative activity at higheffect levels (60 and 75%).

TABLE 15 Inhibition of hCaSMC and hCaEC Cell Proliferation byzotarolimus, paclitaxel and Combinations hCaSMC hCaEC IC₅₀ (nM) IC₅₀(nM) Drug Mean ± SEM Drug Mean ± SEM ABT-578 4.2 ± 1.7 ABT-578 3.6 ± 0.2PAC 3.0 ± 0.5 PAC 4.6 ± 0.3 ABT-578 + 0.1 nM PAC 5.0 ± 0.7 ABT-578 + 1nM PAC 3.6 ± 0.3 ABT-578 + 1 nM PAC 4.0 ± 1.4 ABT-578 + 2.5 nM PAC 2.1(n = 2) ABT-578 + 5 nM PAC N.D.* ABT-578 + 5 nM PAC N.D.* ABT-578 + 10nM PAC N.D.* ABT-578 + 10 nM PAC N.D.* N.D.* Concentrations ofpaclitaxel alone at or above 5 nM inhibit proliferation by greater than50% preventing calculation of ABT-578 IC₅₀'s in these experiments.

These data show that paclitaxel does not block the anti-proliferativeactivity of zotarolimus. Furthermore, high concentrations of zotarolimusand paclitaxel show anti-proliferative activity that appearssynergistic.

EXAMPLE 10 Elution Experiments of Beneficial Agents

Coating the Stents with PC1036

Prior to any experimentation, coated stents were prepared. These were3.0 mm×15 mm 316L electropolished stainless steel stents. Each stent wasspray-coated using a filtered 20 mg/mL solution of phosphoryl cholinepolymer PC1036 (Biocompatibles Ltd.; Farnham, Surrey, UK) in ethanolEtOH). The stents were initially air-dried and then cured at 70° C. for16 hours. They were then sent for gamma irradiation at <25 KGy.

Loading the Stent with Therapeutic Substances

In these experiments, agents were loaded onto stents and elutionprofiles examined. In general, the procedure was as follows. MultiplePC-coated stents were loaded with each drug combination solution. Thesolutions of the drugs were usually in the range of 2-20 mg/mL ofzotarolimus and 1.0-7.0 mg/mL paclitaxel in 100% ethanol, with ˜10%PC1036 added to the solution to enhance film formation. The loading ofdual drug and single drug stents was accomplished by spray loadingappropriate drugs onto a stent in a single pass spray system within anisolator unit. All DES stents were made from Abbott Laboratories TriMaxxN5 construct 15 mm×3.0 mm stents, and all catheters were MedtronicMinneapolis, Minn.) OTW, 15 mm×3.0 mm. The numbers manufactured for eachcombination included units for accelerated elution, drug load content,impurity profile, and animal efficacy testing. The stents were weighedbefore loading with the drug solution. All stents were spray loaded totheir targeted drug contents from solutions including the appropriatedrug(s) and PC1036 in ethanol in a 91:9 ratio. Forpaclitaxel:zotarolimus combinations, stents were prepared at 7 μg/mm ofpaclitaxel with 10 μg/mm of zotarolimus, 3.5 μg/mm of paclitaxel with 5μg/mm of zotarolimus, 1 μg/mm of paclitaxel with 10 μg/mm ofzotarolimus, 7 μg/mm of paclitaxel alone, and 10 μg/mm of zotarolimusalone. Once loaded, all stents were dried in open vials for 30 minutesin an oven set at 40° C. and weighed to determine drug loads. Thedrug-loaded stents were then over-coated with 5 μg/mm of PC1036 byspraying with a 10 mg/ml polymer solution in ethanol.

After over-coating, the stents were cured in an oven at 70° C. for twohours before weighing to determine overcoat weight. After drug loading,the stents were assembled onto catheters, crimped onto the balloon. Thestents were then visually inspected for coating and physical defects.The stent/catheters were inserted into a packaging hoop and thestent/catheter was placed in a Tyvek pouch. The pouch was sealed with aVertrod (San Rafael, Calif.) Impulse Heat sealer. A stent identificationlabel was placed in the bottom corner on the front side of the pouch,outside of the sealed area containing the product. The product was thenplaced in white boxes labeled with the product details and shipped forEtO sterilization. On return from sterilization, the product waspackaged in foil pouches containing sachets of oxygen scavenger anddesiccant. The pouches were labeled with the stent identification numberand product details. The pouches were sealed whilst flushing withnitrogen.

Extracting Drugs from the Stent

For each drug, three stents were used to evaluate the total amount ofdrug loaded. The stents were immersed in 6 mL of 50% acetonitrile, 50%water solution and sonicated for 20 minutes. The concentration of theeach drug in the extraction solution was analyzed by high-pressureliquid chromatography (HPLC).

At the end of the elution experiments discussed below, the stents wereremoved from the elution media and immersed in 6 mL of 50% acetonitrile,50% water solution and sonicated for 20 minutes. The concentration ofeach drug in these vials indicated the amount of the drug remaining onthe stents at the end of the elution experiments.

Elution Process

For assessment of in vitro drug elution, stents (n=3 for each group)were expanded and then placed in a solution of 10 mM acetate buffer(pH=4.0) with 1% Solutol HS 15 BASF; Florham Park, N.Y.) heated to 37°C. in a USP Type II dissolution apparatus. A solubilizing agent wasneeded because the drugs have very low water solubility. The dissolutionmedium was buffered to minimize the degradant of' olimus drugs thathappen at pH's above 6. Buffering at pH 4 solves this problem. Sincethese drugs have minimum dissociation at these pH ranges, pH should havelittle impact on elution rate. Samples were pulled from the dissolutionbath at selected time intervals using a syringe sampler fitted with onlyTeflon, stainless steel or glass surfaces. Aliquots were collected after15 min, 30 min, 1 hr, 2 hr, 4 hr, 6 hr, 8 hr, 12 hr and 24 hr. Thesamples are assayed for zotarolimus and paclitaxel concentration viaHPLC. Data are expressed as drug-eluted in micrograms and mean-percenteluted.

In the HPLC method, it is necessary to use column-switching to minimizeSolutol contamination of the analytical column and to allow rinsing ofthe guard column; otherwise, the system becomes coated with the Solutoland the chromatographic retention changes dramatically. The sample wasfirst injected onto a guard column. Once the analyte peak eluted fromthe guard column and passed onto the analytical column, the guard columnwas switched out of the analytical path. The guard column was thenwashed to remove the Solutol prior to the next injection.

Results

FIGS. 11-13 illustrate the accelerated elution rate of stents loadedwith zotarolimus and paclitaxel at: 7 μg/mm of paclitaxel with 10 μg/mmof zotarolimus, 3.5 μg/mm of paclitaxel with 5 μg/mm of zotarolimus, 1μg/mm of paclitaxel with 10 μg/mm of zotarolimus, 7 μg/mm of paclitaxelalone, and 10 μg/mm of zotarolimus alone onto stents with a 5 μg/mmtopcoat of the polymer PC1036 as detailed above.

In FIG. 11, the 24-hour elution profile shown is where one beneficialagent is paclitaxel and the second beneficial agent is zotarolimus.Elution was carried out as described above. The paclitaxel single drugstent showed a combination of two release profiles, an initially largeburst release (˜60%) followed by a slower, zero-order release rate,whereas dual drug stents that contain both paclitaxel and zotarolimus donot have a burst release.

FIG. 12 presents the same data as FIG. 11, but has been normalized bythe total drug determined on the stent after final stent extract. As canbe seen, 100% of both drugs are recovered from the stent coatings, andthe total drug recovered is in excellent agreement with the drug loadpredicted by stent weight uptake during the drug loading process. Thesedata, along with drug potency and related substances testing on stentsfrom the same batch, indicate that the drugs were stable in the polymercoating when manufactured as described. The small standard deviationsshow that the dual drug elution stents can be manufactured withreproducible elution kinetics.

In FIG. 13, the four curves are the elution profiles (in microgramsreleased versus time) for zotarolimus, alone and in the presence ofpaclitaxel, respectively, under the same conditions. As can be seen, thethree curves that belong to stents with 10 μg/mm of zotarolimus alone orin combination with paclitaxel are very similar. This suggests thatpaclitaxel has little effect on the elution profile of zotarolimus. Thefourth curve (PTX 3.5 and Zotarolimus 5) gave the expected behavior toelute half of the drug as the other stents—as it does.

EXAMPLE 11 Neointimal Formation In Vivo after Stent Implantation

A porcine coronary overstretch model study (Schwartz, 1992) wasconducted to examine neointimal formation for 28 days following stentimplantation. The study evaluated a number of drug-eluting stentsrandomized vs. control zotarolimus-loaded (10 μg/mm; ZoMaxx™) stents.Unexpectedly, the combination of zotarolimus and paclitaxel delivered ona stent is highly efficacious, offering improved reductions inneointimal hyperplasia in the widely utilized porcine coronaryoverstretch model.

A porcine coronary overstretch model study (Schwartz, 1992) wasconducted to examine neointimal formation following stent implantationfor 28 days. The study evaluated a number of drug-eluting stentsrandomized vs. control ZoMaxx™ stents.

Experimental Construct and Methods

In each pig, two major coronary arteries were implanted with one teststent each, and the third major coronary artery was implanted with onezotarolimus (10 μg/mm or 1.69 μg/mm2) coated ZoMaxx™ stent.Additionally, three pigs were implanted with three non-drug containingTriMaxx™ stents (Abbott Laboratories; Abbott Park, Ill.) each (9 totalstents) for comparison. The stents that were compared included ZoMaxx™stents (3.0×15 mm), commercially available sirolimus (8.5 μg/mm or 1.40μg/mm2)-polymer coated Cypher® stents (3.0×13 mm; Cordis Corp.; Miami,Fla.) and paclitaxel-(6.8 μg/mm or 1.0 g/mm2) polymer coated Taxus®stents (3.0×16 mm; Boston Scientific; Natick, Mass.) stents. Theremaining groups of stents were 3.0×15 mm. A paclitaxel stent with thesame drug loading as Taxus (7 μg/mm), but loaded with PC-1036 as thedelivery vehicle, was included in the study PTX-7). In addition, threesets of combination stents were coated that varied in the amounts ofzotarolimus and paclitaxel loaded as shown in Table 16.

TABLE 16 Combination drug-eluting stents used in Example 11 Stentpaclitaxel (μg/mm) zotarolimus (μg/mm) 1 7 10 2 3.5 5 3 1 10

Finally, non-drug eluting TriMaxx stents were included to identify abaseline for neointimal formation.

Stents were implanted with a balloon/artery ratio of 1.30 as determinedby quantitative coronary angiography. There were no cardiac orstent-related mortalities in the study. After 28 days, animals wereeuthanized, and the hearts were removed and perfusion fixed at ˜100 mmHgwith lactated Ringer's solution until cleared of blood followed by 10%neutral buffered formalin. Stented vessels were excised then infiltratedand embedded in methylmethacrylate (MMA). All blocks containing stentedvessels were sectioned so that three in-stent sections plus two controlsections were taken. Two serial thin sections (approximately 5 microns)were taken at each level and stained with Hematoxylin and Eosin (HE) andMasson's Verhoeff Elastin (MVE). Sections were evaluated and scoredusing the BIOQUANT™ TCW98 image analysis system. Average values for allstents within the eight groups for neointimal area, neointimalthickness, and % area stenosis are presented in FIGS. 14-16.

ZoMaxx™, Cypher®, and Taxus® stents had statistically equivalentreductions in formation of neointima as represented by morphometricmeasurements compared to TriMaxx™ stents. The combination stentcontaining 1 μg/mm paclitaxel and 10 μg/mm of zotarolimus PTX/Zot 1/10)showed a significant reduction in neointimal hyperplasia versusTriMaxx™. In addition, the PTX/Zot 1/10 combination stents also showed afurther improvement in reduction in neointima versus ZoMaxx™, Cypher®,and Taxus® stents. Table 17 summarizes the improvements obtained withZoMaxx™ and the PTX/Zot 1/10 combination drug stents versus TriMaxx™.

Each of the state-of-the-art single drug stents, ZoMaxx™, Cypher®, andTaxus® showed dramatic reductions in neointimal formation versusTriMaxx™ controls. For example the average reduction in neointima forZoMaxx™ stents was 34.5% versus control. The PTX/Zot 1/10 combinationstents yielded further improvement in the reduction of neointimalformation over the already impressive results seen with the best singledrug stents available commercially and in clinical trials. The PTX/Zot1/10 combination drug-eluting stents had an average reduction inneointimal hyperplasia of 46.8% when compared to TriMaxx™ non-drugeluting stents. Compared with ZoMaxx™, Cypher®, and Taxus® theadditional dramatic reductions in formation of neointima were 18.8,21.7, and 21.5%, respectively (Table 18).

TABLE 17 Improvements in morphometric measurements versus TriMaxx ®non-drug eluting stents Neointimal Area Neointimal % Area Stent (mm²)Thickness (μm) Stenosis Average ZoMaxx ™ 34.7% 36.0% 32.7% 34.5% PTX/Zot1/10 46.3% 48.5% 45.5% 46.8%

TABLE 18 Improvements in neointimal hyperplasia for paclitaxel (1 μg/mm)and zotarolimus (10 μg/mm) combination drug-eluting stents compared withZoMaxx ™, Cypher ®, and Taxus ® Neointimal Neointimal % Area ComparatorArea Thickness Stenosis Average ZoMaxx ™ 17.8% 19.6% 19.1% 18.8%Cypher ® 24.1% 21.9% 19.2% 21.7% Taxus ® 25.2% 23.2% 16.1% 21.5%

Based on previously published data (Falotico, 2003; Suzuki et al.,2001), one would conclude that combinations of ‘rolimus drugs with asecond drug would offer no advantage. Unexpectedly, when delivered inappropriate combination, the combination of paclitaxel and zotarolimusis highly efficacious, offering improved reductions in neointimalhyperplasia in the widely utilized porcine coronary overstretch model.FIGS. 17 and 18 demonstrate the remarkable difference between ourresults with zotarolimus and paclitaxel PTX/Zot 1/10) and previouslypublished results with sirolimus and dexamethasone (Falotico, 2003;Suzuki et al., 2001). The previous experiment showed no benefit betweenthe combination stent and the single drug-eluting stent. Even with thedramatic improvement in control TriMaxx™ versus BX Velocity®, in theporcine model with the same overstretch ratio, our combination productwas both substantially better than control and substantially andstatistically significantly better than the single drug eluting stent,ZoMaxx™.

The stents coated with 7 μg/mm paclitaxel and 10 μg/mm of zotarolimus(PTX/Zot 7/10) had statistically equivalent reductions in formation ofneointima to ZoMaxx stents (FIGS. 14-16). Consequently, the ideal ratioof paclitaxel derivative to ‘rolimus drug is between 7:10 and 0.1:10 byweight, with the most preferred ratio equal to 1/10. Reduction in thetotal drug dose to 3.5 μg/mm paclitaxel and 5 μg/mm of zotarolimusPTX/Zot 3.5:5) resulted in suboptimal performance, equivalent tonon-drug eluting TriMaxx stents (FIGS. 14-16). Consequently, the optimumtotal dose of paclitaxel derivative and ‘olimus drug should not fallbelow about 150 μg on a 15 mm stent as the ratio of paclitaxelderivative to ‘olimus drug approaches 7:10.

EXAMPLE 12 (prophetic) Clinical Application

The introduction and subsequent widespread use of stents that deliversingle anti-proliferative agents has reduced the restenosis rate to lessthan 10% in the general clinical population. However, a clear rationaleexists for the delivery of appropriate drug combinations from stents totreat patients both in the general clinical population and from avariety of cardiovascular disease subsets to reduce restenosis rates andadverse clinical events still further. For example, it is well acceptedthat the rate of restenosis is significantly increased in stenteddiabetic patients when compared to those without the disease, and thatan inflammatory response to stenting exists in both diabetic andnon-diabetic patients (Aggarwal et al., 2003). In addition, inflammationis a hallmark in patients with acute coronary syndrome (ACS), a termwhich defines a range of acute myocardial ischemic conditions, includingunstable angina, non-ST segment elevation myocardial infarction, as wellas infarction associated with persistent ST-segment elevation. Thesepatients are often prime candidates for stent deployment, and relativeto the general patient population undergoing percutaneous interventionPCI), have significantly higher rates of recurrent ischemia,reinfarction and subsequent need for repeat PCI procedures. Finally,obesity is often associated with a pro-inflammatory state andendothelial dysfunction. Both conditions are known to be independentpredictors of early restenosis after coronary stent placement. In fact,a case has been made for an association between obesity, interleukin-6(IL-6) production by adipocytes and coronary artery disease, suggestinga link between elevations of this inflammatory cytokine and thedevelopment of CAD in this sub-set of patients QYudkin et al., 2000).

Diabetic patients are known to exhibit higher levels of the inflammatorymarker, c-reactive protein (CRP) than non-diabetic patients (Aggarwal etal., 2003; Dandona and Aljada, 2002). This protein has been clearlyidentified as a key inflammatory mediator in patients with coronaryartery disease, and is a predictor of adverse events in patients withsevere unstable angina (Biondi-Zoccai et al., 2003). CRP is known tostimulate the production of monocyte chemoattractant protein (MCP-1) byhuman endothelial cells. The release of this mediator is accompanied bythe influx of monocytes, resulting in a marked inflammatory state asthese cells are activated and move into the sub-endothelial space, wherethey form foam cells containing oxidized low-density lipoprotein LDL).Plasma IL-6 and tumor necrosis factor-α TNF-α) are inflammatorycytokines that are also elevated in the obese patient, and in type 2diabetics. In fact, elevation of high-sensitivity CRP, IL-6 or serumvascular cell adhesion molecule-1 (VCAM-1) have been associated withincreased mortality in patients with coronary artery diseases (Roffi andTopol, 2004). Since it has been shown that neointimal formation, ahallmark of the restenotic process, is accentuated by inflammation, theuse of stents which deliver a combination of agents withanti-inflammatory and antiproliferative activities such as paclitaxeland zotarolimus to the local vessel environment would be expected tohave clear utility in diabetic patients.

Disruption of an atheromatous plaque is central to the initiation of anacute coronary syndrome (Grech and Ramsdale, 2003). Plaque rupture maybe induced by increased concentrations of matrix metalloproteinasessecreted by foam cells, leading to plaque instability and ultimaterupture of the thin fibrous cap which overlies the developing lesion. Inaddition, tissue factor, which is expressed on the surface of foamcells, activates coagulation factor VII, which leads to the formation ofthrombin. Generation of this protein leads to platelet activation andaggregation, as well as the conversion of fibrinogen to fibrin, and theclear formation of thrombus. Initial concern regarding the deployment ofstents in this setting appears unfounded, since improvements in stentdeployment and technique have shown that stented patients have lessrecurrent ischemia, reinfarction and need for repeat angioplasty (Grechand Ramsdale, 2003). The close relationship between inflammation and thedevelopment of coronary artery lesions makes the use of stents thatdeliver a combination of agents with anti-inflammatory andanti-proliferative activities such as paclitaxel and zotarolimus to thelocal vessel environment an attractive approach to treating suchpatients.

The stents described herein will be deployed in patients who arediagnosed with ischemic heart disease due to stenotic lesions incoronary arteries and in subsets of the clinical population at higherrisk for recurrent coronary disease and other adverse clinical events.Other targets for intervention include peripheral vascular diseases suchas stenoses in the superficial femoral arteries, renal arteries, iliacs,and vessels below the knee. Target vessels for interventional procedureswill be reached using percutaneous vascular access via either thefemoral or radial artery, and a guiding catheter will be inserted intothe vessel. The target lesion will then be crossed with a guide wire,and the balloon catheter will be inserted either over the wire or usinga rapid exchange system. The physician will determine the appropriatesize of the stent to be implanted by online quantitative coronaryangiography (QCA) or by visual estimate. The stent will be deployedusing appropriate pressure as indicated by the compliance of the stent,and a post-procedure angiogram can then be obtained. When the procedureis completed, the patient will be regularly monitored for angina statusand for the existence of any adverse events. The need for repeatprocedures will also be assessed.

It is understood that the foregoing detailed description andaccompanying examples are merely illustrative and are not to be taken aslimitations upon the scope of the invention, which is defined solely bythe appended claims and their equivalents. Various changes andmodifications to the disclosed embodiments will be apparent to thoseskilled in the art. Such changes and modifications, including withoutlimitation those relating to the chemical structures, substitutents,derivatives, intermediates, syntheses, formulations and/or methods ofuse of the invention, may be made without departing from the spirit andscope thereof. //

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1. A drug delivery medical device, comprising: a medical devicecomprising a pharmaceutically acceptable carrier or excipient; and ataxane or its derivatives, prodrugs, or salts and rapamycin or itsderivatives, prodrugs, or salts associated with the medical device;wherein the ratio, r, by weight of the taxane or its derivatives,prodrugs, or salts to rapamycin or its derivatives, prodrugs, or saltsis 7:10≧r≧0.01:10; and wherein neointimal hyperplasia is reduced whenthe medical device is implanted in a lumen of a blood vessel of asubject when compared to a control system.
 2. The drug delivery medicaldevice according to claim 1, wherein neointimal hyperplasia is reduced≧10% when compared to the control system.
 3. The drug delivery medicaldevice according to claim 1, wherein neointimal hyperplasia is reduced≧15% when compared to the control system.
 4. The drug delivery medicaldevice according to claim 1, wherein neointimal hyperplasia is reduced≧20% when compared to the control system.
 5. The drug delivery medicaldevice according to claim 2, 3, or 4, wherein neointimal hyperplasia ismeasured by at least one technique selected from the group consisting ofneointimal area measurements, neointimal thickness measurements andpercent area stenosis measurements.
 6. The drug delivery medical deviceaccording to claim 1, wherein the subject is a human being.
 7. The drugdelivery medical device according to claim 1, wherein the taxane ispaclitaxel.
 8. The drug delivery medical device according to claim 1,wherein r=1:10.
 9. The drug delivery medical device according to claim1, wherein the ratio, r, exerts an additive effect.
 10. The drugdelivery medical device according to claim 9, wherein r=1:10.
 11. Thedrug delivery medical device according to claim 1, wherein the medicaldevice comprises a stent.
 12. A medical device for providing controlledrelease delivery of drugs for treating or inhibiting neointimalhyperplasia in a blood vessel, comprising: a taxane or salts, prodrugs,or derivatives thereof; and rapamycin, or salts, prodrugs, orderivatives thereof; wherein the taxane or its salts, prodrugs, orderivatives complements the activity of the rapamycin or its salts,prodrugs, or derivatives, and the rapamycin or its salts, prodrugs, orderivatives complements the activity of the taxane or its salts,prodrugs, or derivatives; and wherein the ratio, r, of the taxane or itssalts, prodrugs, or derivatives to the rapamycin or its salts, prodrugs,or derivatives by weight is 7:10≧r≧0.01:10.
 13. The medical deviceaccording to claim 12, with the proviso that the rapamycin analog is notzotarolimus.
 14. The medical device according to claim 12, wherein themedical device is a stent.
 15. The medical device according to claim 14,wherein the stent further comprises a coating on a surface.
 16. Themedical device according to claim 15, wherein the coating comprises apolymer.
 17. The medical device according to claim 16, wherein thepolymer comprises a phosphorylcholine polymer.
 18. The medical deviceaccording to claim 16, wherein the polymer is selected from the groupconsisting of fluoropolymers, polyacrylates, silicones, resins, nylons,and poly(amides).
 19. The medical device according to claim 15, whereinthe taxane or its salts, prodrugs, or derivatives and the rapamycin orits salts, prodrugs, or derivatives are associated with the coating. 20.The medical device according to claim 12, the ratio, r, exerts anadditive effect.
 21. The system according to claim 12, wherein r=1:10.